Compositions and Methods for Modulating an Immune Response

ABSTRACT

The invention provides methods of modulating follicular regulatory T (TFR) cell-mediated immune responses, follicular helper T (TFH) cell-mediated immune responses or both, and the use of those methods in the treatment of diseases or conditions mediated by TFR or TFH cells. The invention also provides novel methods for identifying TFR and TFH cells in a population of cells. The invention also provides compositions comprising TFR cells that have enhanced suppressive activity as compared wild type TFR cells. The invention also provides compositions comprising T follicular regulatory (TFR) cells isolated from the peripheral blood of a subject wherein the composition is enriched for TFR cells. Methods of making and using the compositions of the invention to modulate an immune response are also provided.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/707,596, filed May 8, 2015 which is a continuation ofPCT/US2013/069197, filed Nov. 8, 2013, which claims the benefit of U.S.Provisional Application No. 61/724,424, filed on Nov. 9, 2012. Theentire teachings of the above applications are incorporated herein byreference.

GOVERNMENT SUPPORT

This invention was made with government support under grant numbers T32AI070085; R01 AI40614, P01 78897 and T32 HL007627 awarded by TheNational Institutes of Health. The Government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

Regulation of immune responses is central for the prevention ofinflammatory and autoimmune disorders. While downregulation of theimmune system can be achieved by way of immunosuppressive therapy,agents that generally suppress the immune system leave subjectssusceptible to other disorders, including infections and cancers. Ameans for controlling the aberrant activation of an immune response tospecific antigens would be a major advance in the treatment ofautoimmune disorders, graft versus host disease and the side effects ofgene therapy, as it would allow downregulation of the immune responseagainst a particular target antigen, but would otherwise leave theimmune system functional against invading pathogens and tumor associatedantigens. Conversely, methods of specifically improving immunogenicityof specific antigens to which immune responses are desired would be oftremendous benefit in promoting desired immune responses; for example inthe context of vaccination and promoting responsiveness to antigensincluding tumor antigens.

T helper (Th) cells are a class of CD4+ cells that function to regulatethe proliferation of B cells and B cell responses. Th cells play animportance role in humoral immunity and immunopathology. Follicularhelper T cells (TFH) are a recently defined subset of CD4+ T cells thatare essential for helping cognate B cells form and maintain the germinalcenter (GC) reaction, and for development of humoral immune responses.These cells are universally defined by expression of the chemokinereceptor CXCR5, which directs them to the B cell follicles via gradientsof the chemokine CXCL13¹. TFH cells also express the transcriptionfactor Bcl6 (which represses Blimp-1/Prdm1) and high levels of thecostimulatory receptor ICOS, which are both critical for theirdifferentiation and maintenance¹⁻⁴. In addition, TFH cells secrete largeamounts of IL-21, which aids in GC formation, isotype switching andplasma cell formation⁵. In humans and mice functionally similar TFHcells can be found in secondary lymphoid organs. CXCR5⁺ TFH cells arealso present in peripheral blood and seen at elevated levels inindividuals with autoantibodies, including systemic lupus erythematosis,myasthenia gravis and juvenile dermatomyositis patients. However, thefunction of these circulating TFH remains unclear⁶⁻⁹.

Regulatory T cells (Tregs) have pluripotent anti-inflammatory effects onmultiple cell types. In particular, they control the activation ofinnate and adaptive immune cells. Tregs acting in an antigen-specificmanner reduce effector T cell activation and function, for example,after effector T cells have successfully mounted an attack against aninvading pathogen, or to suppress reactivity to self-antigen and therebyprevent autoimmune disease.

Two subsets of Tregs are classified according to the location at whichthey develop in vivo. Naturally occurring Tregs (nTreg) develop in thethymus and suppress self-reactive immune responses in the periphery,whereas adaptive Tregs (aTreg) develop in the periphery fromconventional CD4⁺ T cells to ensure tolerance to harmless antigens,including those derived from, for example, food and intestinal flora.Both subsets of Treg cells are characterized by expression of highlevels of CD25 and the transcription factor Foxp3. Tregs are thought toinhibit the antigen-specific expansion and/or activation ofself-reactive effector T cells and to secrete suppressive cytokines,including TGF or IL-10. Because of their potential to provideantigen-specific immune regulation without generalizedimmunosuppression, Tregs have been contemplated for use in cell-basedtherapy for inflammatory or autoimmune disorders.

T follicular regulatory (TFR) cells are newly defined subset ofCD4⁺CXCR5⁺ cells which are positive for the transcription factors FoxP3,Bcl6 and Prdm1/Blimp1 and function to inhibit the germinal centerresponse²¹⁻²³.

PD-1 has been identified as a receptor which binds to PD-L1 and PD-L2.PD-1 is a member of the immunoglobulin gene superfamily. PD-1 (Ishida etal. (1992) EMBO J. 11:3887; Shinohara et al. (1994) Genomics 23:704;U.S. Pat. No. 5,698,520) has an extracellular region containingimmunoglobulin superfamily domain, a transmembrane domain, and anintracellular region including an immunoreceptor tyrosine-basedinhibitory motif (ITIM). PD-1 transmits a negative signal to immunecells, similar to CTLA4. PD-1 ligand proteins are expressed on thesurface of antigen presenting cells, and other cell types; and canprovide a costimulatory signal to immune cells or can transmitdownmodulatory signals to immune cells, depending upon the protein towhich they bind. While transmission of an inhibitory signal leads todownmodulation in immune cell responses (and a resulting downmodulationin the overall immune response), the prevention of an inhibitory signal(e.g., by using a non-activating antibody against PD-1) in immune cellsleads to upmodulation of immune cell responses (and a resultingupmodulation of an immune response).

TFH cells express high levels of programmed death (PD) 1 receptor(CD279). Signaling through PD-1 attenuates TCR signals and inhibits Tcell expansion, cytokine production and cytolytic function. In addition,PD-1 promotes the development of induced regulatory T (iTreg) cells fromnaïve lymphocytes¹⁰⁻¹⁴. PD-1 has two ligands, PD-L1 (B7-H1) and PD-L2(B7-DC). PD-L1 is more widely expressed than PD-L2, but PD-L1 and PD-L2both can be expressed on GC B cells and dendritic cells¹⁵. Perturbationstudies suggest critical roles for this pathway in regulating humoralimmune responses. However, there are conflicting reports as to thefunction of the PD-1 pathway in controlling humoral immunity. Somestudies have found that humoral responses are attenuated¹⁶⁻¹⁸, whileothers have seen that humoral responses are heightened^(19, 20) whenPD-1:PD-L interactions are prevented.

PD-1 also is found on TFR cells. These cells originate from naturalregulatory T cell precursors, but express similar levels of ICOS, CXCR5and PD-1 as TFH cells. Since ICOS, CXCR5 and PD-1 have been widely usedto identify and purify ‘TFH cells’, it seems likely that the inabilityto define clear functions for PD-1 in GC responses derives fromexperimental systems containing mixtures of stimulatory TFH cells andinhibitory TFR cells.

The present inventors have discovered that PD-1:PD-L1 interactions limitTFR cell differentiation and function. This discovery has elucidatednovel approaches to modulating an immune response for use in therapy.

This discovery has also elucidated novel cell markers for identifyingand separating TFR and TFH cells from all other cell types. Thesemarkers are also useful for selectively modifying TFR-mediated and/orTFH mediated immune responses in vivo and in vitro.

SUMMARY OF THE INVENTION

The invention is based, in part on the discovery that PD-1 regulatesimmune responses by inhibiting differentiation and function of TFR cellsin both lymph nodes and blood. This discovery has led to strategies formodulating an immune response in vivo and in vitro by altering theinteraction of PD-1 receptors on TFR cells with PD-1 ligands.

The invention is also based in part on the discovery of cell markers onTFH and THR cells that enable such cells to be more easily identified,distinguished and tracked in vivo and/or separated into highly purifiedhomogenous cell populations of TFH cells or TFR cells. Such markers makeit possible to add specificity and reliability in detecting TFR or TFHcells either in situ, in circulation, or as disseminated cells which isparticularly useful for example in monitoring disease burden and intracking the responses of TFH cells and TFR cells to particular stimulisuch as agents intended to modulate TFH and TFR cell-mediated immuneresponses. These markers are also useful for selectively modifying TFRcell-mediated, and/or TFH cell-mediated immune responses in vivo and invitro.

In one embodiment the invention provides TFR cells having enhancedsuppressive activity as compared to wild type TFR cells. In oneembodiment, the invention provides compositions comprising TFR cellshaving enhanced suppressive activity. TFR cells having enhancedsuppressive activity as compared to wild type TFR cells arecharacterized by their greater capacity for antibody inhibition ascompared to WT TFR, as measured by an in vivo or in vitro assays forantibody suppression. In one embodiment, the enhanced TFR cells have anincrease in capacity of at least 2 fold to inhibit antibody productionas compared to WT TFR cells.

In one embodiment the invention provides methods for generating TFRcells having enhanced suppressive activity. In accordance with theinvention, TFR cells having enhanced suppressive activity may beprepared, ex vivo, from a starting population of cells which compriseTFR cells and/or TFR precursor cells such as FoxP3+T regulatory (Treg)cells, isolated from the blood, tissues or organs of one or moresubjects. The starting population of cells may optionally be sortedbased on surface markers for Tcells and Tcell subsets. The startingpopulation of cells is expanded and/or activated in the presence of aPD-1 receptor antagonist or PD-1 ligand inhibitor. Theexpanded/activated cells may optionally be sorted based on surfacemarkers for T cells and T cell subsets to obtain a composition of cellsenriched for a particular T cell subset (e.g. TFR cells or TFH cells).

The present inventors have also discovered that TFR cells derived fromthe peripheral blood of a subject are potent inhibitors of TFH mediatedantibody production but do no inhibit other arms of the immune system.Therefore, the invention provides compositions comprising TFR cellsisolated from the peripheral blood of a subject. The compositions may beenriched for the peripheral blood TFR cells by purifying TFR cells fromother PMBCs and optionally sorting for TFR cells based on surfacemarkers. Compositions of TFR cells derived from peripheral blood mayalso be expanded and/or activated to produce a clonal population of TFRcells based on the original population of TFR cells derived from theperipheral blood of the patient.

Compositions comprising TFH cells which are also present in theperipheral blood of a subject may be provided in the same manner. Theinventors have discovered that such compositions, particularly whenenriched for TFH cells, are capable of rapidly upregulating an antibodyresponse in vitro and in vivo.

The inventors have also discovered that increasing the ratio of TFRcells to TFH cells in a subject prior to, or during an immune responseby the subject, inhibits antibody production. Therefore, the inventionprovides a method for suppressing an immune response in a subjectwherein suppression of an immune response is desired, comprisingincreasing the ratio of TFR cells to TFH cells by administering acomposition enriched with TFR cells derived from the peripheral blood ofa subject (or an expanded/activated population thereof) or administeringa composition comprising TFR cells having enhanced suppressive activity.

The invention also provides methods of promoting rapid antibodyproduction in a subject comprising increasing the number of TFH cells ina patient by administering a composition enriched with TFH cells derivedfrom the peripheral blood of a subject (or an expanded/activatedpopulation thereof).

The compositions of TFR cells and TFH cells of the invention are alsouseful as adjuvants as a part of a vaccination regimen. When used inthis manner, the compositions enhance the efficacy of such vaccines.

The invention further provides compositions and methods for suppressingpathogenic antibody responses in a patient comprising selectivelymodulating differentially expressed receptors on TFR cells, TFH cells,or both TFR cells and TFH cells with agents capable of modulating suchreceptors in amounts effective to suppress a pathogenic antibodyresponse.

The invention further provides compositions and methods for enhancing aprotective antibody response in a patient comprising selectivelymodulating differentially expressed receptors on TFR cells, TFH cells,or both TFR cells and TFH cells with agents capable of modulating suchreceptors in amounts effective to enhance a protective antibody responsein a patient.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention, as illustrated inthe accompanying drawings in which like reference characters refer tothe same parts throughout the different views. The drawings are notnecessarily to scale, emphasis instead being placed upon illustratingthe principles of the invention.

FIG. 1A. PD-1 signaling in FoxP3 Tregs limits the generation of Tfollicular regulatory cells. Quantitation of TFR cells. WT mice wereimmunized with MOG/CFA and 7 days later draining lymph nodes wereisolated and immediately stained for CD4⁺FoxP3⁺ICOS⁺CXCR5⁺CD19⁻ Tfollicular regulatory cells (TFR), CD4⁺FoxP3⁻ICOS⁺CXCR5⁺CD19⁻ Tfollicular helper cells (TFH), CD4⁺ICOS⁻CXCR5⁻CD19⁻ cells (naive) orCD4⁺ICOS⁺CXCR5⁻CD19⁻ cells (ICOS+). Numbers indicate percentages ofcells located within each gate.

FIG. 1B. PD-1 expression by flow cytometry on WT naive, ICOS+, TFR andTFH cells. Populations were gated as in FIG. 1A. Data represent means of5 mice per group. All error bars indicate standard error

FIG. 1C Gating of TFR cells from total FoxP3⁺ cells in WT and PD-1^(−/−)mice immunized with MOG/CFA and analyzed 7 days later and stained as inFIG. 1A.

FIG. 1D. Quantitation of WT or PD-1^(−/−) TFR cells gated in FIG. 1C andexpressed as a percentage of CD4⁺FoxP3⁺ (left), percentage of total CD4T cells (middle), or percentage of CD4⁺ICOS⁺CXCR5⁺CD19⁻ gate (right).Data represent means of 5 mice per group. All error bars indicatestandard error

FIG. 1E. Quantitation of TFH cells as a percentage of total CD4 T cells.Data represent means of 5 mice per group. All error bars indicatestandard error.

FIG. 1F. PD-1 on FoxP3⁺ cells has a cell-intrinsic role in inhibitingTFR differentiation in vivo. Schematic design of a transfer assay inwhich 2D2 TCR transgenic CD4⁺FoxP3⁺CXCR5⁻ non-TFR Tregs were transferredinto WT mice which were subsequently immunized with MOG/CFA. Draininglymph nodes were harvested 7 days later and analyzed for TFR cells.

FIG. 1G. representative gating of TFR cells from transfer experimentsdescribed in FIG. 1F.

FIG. 1H. Quantitation of TFR cells from transfer experiments expressedas a percentage of FoxP3 GFP⁺ cells present on day 7 post immunizationper lymph node. All data are representative of at least two independentexperiments with at least 5 mice per group. All error bars indicatestandard error. * P<0.05, ** P<0.005.

FIG. 1I. Quantitation of TFR cells from transfer experiments expressedas total cell number per lymph node. All data are representative of atleast two independent experiments with at least 5 mice per group. Allerror bars indicate standard error. * P<0.05, ** P<0.005

FIG. 2A. PD-1 deficient TFR cells have altered expression of activationmarkers. TFR cell gating strategy. WT or PD-1^(−/−) mice were immunizedwith MOG/CFA and draining lymph nodes were harvested 7 days later.

FIG. 2B. CD25 expression on WT and PD-1^(−/−) CD4 subsets gated as inFIG. 2A. Overlay histograms of WT and PD-1^(−/−) TFR cells (left) andmean fluorescence intensity (MFI) in CD4 subsets gated as in FIG. 1A(right). Data represent means of 5 mice per group.

FIG. 2C. CD69 expression on WT and PD-1^(−/−) CD4 subsets gated as inFIG. 2A. Overlay histograms of WT and PD-1^(−/−) TFR cells (left) andMFI (right). Data represent means of 5 mice per group.

FIG. 2D. Intracellular staining of cell cycle marker Ki67 in populationsas in (a). Overlay histograms of WT and PD-1^(−/−) TFR cells (left) andpercent Ki67 high (right) in CD4 subsets gated as in FIG. 2A. Ki67 highwas defined as the highest intensity peak on WT TFR cells and is denotedby a black bar on the histogram. Data represent means of 5 mice pergroup. All data are representative of at least two independentexperiments. All error bars indicate standard error. * P<0.05, **P<0.005, *** P<0.0005.

FIG. 3A. PD-1 deficient TFR cells are capable of homing to germinalcenters (GCs). Micrographs of draining lymph node sections from WT miceimmunized with MOG/CFA and harvested 7 days later. Sections were cut andstained for GL-7 (green), PNA (red) and IgD (blue). GCs were identifiedby PNA and GL7 positive, but IgD negative, staining. GCs are indicatedwith a white dotted line.

FIG. 3B. Ki67 staining in GCs. Sections were stained for the cell cyclemarker Ki67 (blue), FoxP3 (red) and GL7 (green).

FIG. 3C. Colocalization of CD4 and FoxP3. Sections were stained for CD4(blue), FoxP3 (red) and GL7 (green). Box indicates magnificationhighlighting CD4 positive staining on FoxP3⁺ cells.

FIG. 3D. Colocalization of FoxP3 in the nucleus. Sections were stainedwith the nuclear stain DAPI (blue), FoxP3 (red) and GL7 (green). Boxindicates magnification highlighting FoxP3 protein within DAPI positivenuclei.

FIG. 3E. Comparison of FoxP3⁺ TFR cells in germinal centers of WT andPD-1^(−/−) mice. Representative GC staining in WT and PD-1^(−/−) lymphnodes 7 days after immunization with MOG/CFA.

FIG. 3F. Average GC area was determined by calculating the area withinthe dotted lines according to materials and methods. Data represent meanarea per lymph node of 5 individual mice.

FIG. 3G. Numbers of FoxP3⁺ cells contained within GCs. Data representmean per GC from 5 pooled mice.

FIG. 3H. Quantitation of the distance of each FoxP3⁺ cell to the GCborder. The distance for each FoxP3⁺ cell in FIG. 3E from the GC borders(dotted line in FIG. 3E) was calculated as described in materials andmethods.

FIG. 3I CXCR5 expression was quantified on WT and PD-1^(−/−)CD4⁺ICOS⁺CXCR5⁺FoxP3⁻CD19⁻ TFH and CD4⁺ICOS⁺CXCR5⁺FoxP3⁺CD19⁻ TFR cellsby flow cytometry 7 days after MOG/CFA immunization. Data representmeans of 5 mice per group. * P<0.05, ** P<0.005, *** P<0.0005.

FIG. 4A. PD-1 deficient TFR cells have enhanced regulatory capacity. TFRcells express high levels of GITR. WT mice were immunized with MOG/CFAand 7 days later lymph node cells were isolated and expression of GITRon TFR (CD4⁺FoxP3⁺ICOS⁺CXCR5⁺CD19⁻, blue) and TFH(CD4⁺FoxP3⁻ICOS⁺CXCR5⁺CD19⁻, red) was quantified as shown by histogramoverlays.

FIG. 4B. Expression of FoxP3 mRNA in sorted TFR(CD4⁺GITR⁺ICOS⁺CXCR5⁺CD19⁻), TFH (CD4⁺GITR⁻ICOS⁺CXCR5⁺CD19⁻) and naive(CD4⁺ICOS⁻CXCR5⁻CD19⁻) cells. Data represent fold change in mRNAnormalized to Hprt.

FIG. 4C. Bcl6 expression analyzed by intracellular flow cytometry on TFHand TFR cells from WT (blue) and PD-1^(−/−) (green) mice. Data representmeans from at least three separate experiments in which cells weresorted from lymph nodes of 10 pooled mice.

FIG. 4D. mRNA expression of blimp-1/Prdm1 from sorted WT (blue) andPD-1^(−/−) (green) TFR and TFH cells and in CD4-ICOS⁻CXCR5⁻ (naive)cells quantified by qPCR analysis. Data represent means from at leastthree separate experiments in which cells were sorted from lymph nodesof 10 pooled mice.

FIG. 4E. mRNA expression of Rorc from sorted WT (blue) and PD-1^(−/−)(green) TFR and TFH cells and in CD4−ICOS⁻CXCR5⁻ (naive) cellsquantified by qPCR analysis. Data represent means from at least threeseparate experiments in which cells were sorted from lymph nodes of 10pooled mice.

FIG. 4F. mRNA expression of Irf4 from sorted WT (blue) and PD-1^(−/−)(green) TFR and TFH cells and in CD4−ICOS⁻CXCR5⁻ (naive) cellsquantified by qPCR analysis. Data represent means from at least threeseparate experiments in which cells were sorted from lymph nodes of 10pooled mice.

FIG. 4G. Design of assay to analyze capacity of TFR cells to inhibitactivation of naïve CD4 T cells. WT and PD-1^(−/−) mice were immunizedwith MOG/CFA and TFR cells were sorted from draining lymph nodes andplated 1:1:1 with CF SE-labeled CD4 naïve WT (CD4⁺CD62L⁺FoxP3⁻)responder cells and WT GL7⁻B220⁺ B cells from MOG/CFA immunized micealong with anti-CD3 and anti-IgM for 4 days. 3 days later samples wereanalyzed by flow cytometry.

FIG. 4H. TFR cells suppress activation of naïve T cells to a greaterextent than WT TFR cells. T responders from suppression assays from FIG.4G were analyzed for CD69 expression by measuring CFSE dilution. %divided indicates percent of cells that have gone through at least onedivision.

FIG. 4I. PD-1^(−/−) TFR cells suppress activation of naïve T cells to agreater extent than WT TFR cells. T responders from suppression assaysfrom FIG. 4G were analyzed for CD69 proliferation by measuring CFSEdilution. % divided indicates percent of cells that have gone through atleast one division.

FIG. 4J. In vitro IgG suppression assay design. TFR cells sorted as inFIG. 4G were plated in a 1:1:1 ratio of TFR (CD4⁺ICOS⁺CXCR5⁺GITR⁺CD19⁻),TFH (CD4⁺ICOS⁺CXCR5⁺GITR⁻CD19⁻), and B (GL-7⁻B220⁺) cells from draininglymph nodes of MOG/CFA immunized mice in the presence of anti-CD3 andanti-IgM for 6 days. Total IgG was measured by ELISA from supernatants.

FIG. 4K. Suppression assay using two concentrations of anti-CD3.

FIG. 4L. PD-1 deficient TFR cells suppress IgG production to a greaterextent than WT TFR cells at a 1:1 TFR:TFH ratio. Naive(CD4⁺ICOS⁻CXCR5⁻CD19⁻) cells from immunized mice were included ascontrols. Data indicates means+/−standard error of replicate wells andis representative of at least two experiments. * P<0.05, ** P<0.005, ***P<0.0005.

FIG. 4M. PD-1 deficient TFR cells suppress IgG production to a greaterextent than WT TFR cells at a 1:5 TFR:TFH ratio. Data indicatesmeans+/−standard error of replicate wells and is representative of atleast two experiments. * P<0.05, ** P<0.005, *** P<0.0005.

FIG. 5A. PD-1 controls circulating blood TFR cells. Gating strategy toidentify circulating TFH and TFR cells from blood. WT mice wereimmunized with MOG/CFA and blood was collected 7 days later by cardiacpuncture. TFH and TFR populations were gated as shown.

FIG. 5B. Quantitation of blood TFH and TFR cells following MOG/CFAimmunization. Mice were immunized as in FIG. 5A and sacrificed on theindicated days. Blood was collected and cells stained and gated as inFIG. 5A.

FIG. 5C. Ki67 expression in blood and lymph node TFH, TFR and naïve(CD4⁺ICOS⁻CXCR5⁻) cells 7 days after MOG/CFA immunization. All dataindicates means+/−standard error of 5 mice and is representative of atleast two independent experiments. * P<0.05, ** P<0.005, *** P<0.0005.

FIG. 5D. Comparison of blood TFH and TFR cells in WT and PD-1^(−/−) miceimmunized as in FIG. 5A and harvested 7 days after immunization. BloodTFH cells are shown gated on FoxP3⁻CD4⁺CD19⁻ (left) and TFR cells areshown gated on FoxP3⁺CD4⁺CD19⁻ (right).

FIG. 5E. Comparison of blood TFH and TFR cells in WT and PD-1^(−/−) miceimmunized as in FIG. 5A and harvested 7 days after immunization.Quantitation of blood TFH and TFR cells from immunized WT and PD-1^(−/−)mice gated as in FIG. 5D and expressed as a percent of CD4⁺CD19⁻ cells.All data indicates means+/−standard error of 5 mice and isrepresentative of at least two independent experiments. * P<0.05, **P<0.005, *** P<0.0005.

FIG. 5F. Comparison of blood TFH and TFR cells in WT and PD-1^(−/−) miceimmunized as in FIG. 5A and harvested 7 days after immunization.Quantitation of CXCR5⁻ FoxP3⁺ cells from immunized WT and PD-1^(−/−)mice, expressed as a percentage of CXCR5⁻ CD4⁺ cells. All data indicatesmeans+/−standard error of 5 mice and is representative of at least twoindependent experiments. * P<0.05, ** P<0.005, *** P<0.0005.

FIG. 6A. PD-L1 but not PD-L2 controls blood TFR cells. PD-L1 and PD-L2expression on B cell subsets. WT mice were immunized with NP-OVAsubcutaneously and 12 days later germinal center B (GC B), GL7⁻, andplasma cells (PC) from draining lymph nodes were analyzed for PD-L1(top) and PD-L2 (bottom) expression.

FIG. 6B. PD-L1 and PD-L2 expression on dendritic cells (DC). WT micewere immunized with NP-OVA and 3 days later CD8α⁺ DC and CD8α⁻ DCsubsets from draining lymph nodes were analyzed for PD-L1 (top) andPD-L2 (bottom) expression.

FIG. 6C. Lymph node and blood TFH and TFR cells in PD-1 ligand deficientmice. WT, PD-L1^(−/−) and PD-L2^(−/−) mice were immunized with MOG/CFA,and 7 days later draining lymph nodes and blood were harvested andanalyzed for TFH CD4 T cells. Data represent means of 5 mice per group.All data are representative of at least two independent experiments. *P<0.05, ** P<0.005, *** P<0.0005.

FIG. 6D. Lymph node and blood TFH and TFR cells in PD-1 ligand deficientmice. WT, PD-L1^(−/−) and PD-L2^(−/−) mice were immunized with MOG/CFA,and 7 days later draining lymph nodes and blood were harvested andanalyzed for TFR CD4 T cells. Data represent means of 5 mice per group.All data are representative of at least two independent experiments. *P<0.05, ** P<0.005, *** P<0.0005.

FIG. 6E. Lymph node and blood TFH and TFR cells in PD-1 ligand deficientmice. WT, PD-L1^(−/−) and PD-L2^(−/−) mice were immunized with MOG/CFA,and 7 days later draining lymph nodes and blood were harvested andanalyzed for CXCR5⁻ FoxP3⁺ CD4 T cells. Data represent means of 5 miceper group. All data are representative of at least two independentexperiments. * P<0.05, ** P<0.005, *** P<0.0005.

FIG. 7A. Blood TFR cells require ICOS and CD28 costimulation. TFH andTFR gating in WT and ICOS^(−/−) mice. Mice were immunized with MOG/CFAand 7 days later draining lymph nodes (dLN) and blood were harvested.TFH cells were gated as CD4⁺CD44⁺CXCR5⁺FoxP3⁻CD19⁻, and TFR cells asCD4⁺CD44⁺CXCR5⁺FoxP3⁺CD19⁻ cells.

FIG. 7B TFH quantitation in lymph nodes (dLN) and blood of WT andICOS^(−/−) mice as in FIG. 7A.

FIG. 7C TFR quantitation in lymph nodes (dLN) and blood of WT andICOS^(−/−) mice as in FIG. 7A.

FIG. 7D. TFH and TFR gating strategy in WT and CD28^(−/−) mice. Micewere immunized as in FIG. 7A and TFH cells were gated asCD4⁺ICOS⁺CXCR5⁺FoxP3⁻ CD19⁻ and TFR cells as CD4⁺ICOS⁺CXCR5⁺FoxP3⁺CD19⁻.

FIG. 7E. TFH quantitation in lymph nodes and blood of WT and CD28^(−/−)mice gated as in FIG. 7D. All data are representative of at least twoindependent experiments. * P<0.05, ** P<0.005, *** P<0.0005.

FIG. 7F. TFR quantitation in lymph nodes and blood of WT and CD28^(−/−)mice gated as in FIG. 7D. All data are representative of at least twoindependent experiments. * P<0.05, ** P<0.005, *** P<0.0005.

FIG. 8A. PD-1 deficient blood TFR cells more potently regulate antibodyproduction in vivo. Experimental strategy to assess blood TFH and TFRcell function by transfer of blood TFH and/or TFR cells into mice thatlack both lymph node and blood TFH/TFR cells. Blood TFH and/or TFR cellswere isolated from 20 pooled mice immunized with NP-OVA 8 dayspreviously and CD4⁺CXCR5⁺GITR⁻CD19⁻ TFH and CD4⁺CXCR5⁺GITR⁺CD19⁻ TFRcells were purified by cell sorting; recipient CD28^(−/−) or TCRα^(−/−)mice received either no cells, 4×10⁴ TFH cells, or 4×10⁴ TFH plus 2×10⁴TFR cells. One day later recipients were immunized with NP-OVA. 12 dayslater sera were collected and NP-specific antibody titers quantified byELISA.

FIG. 8B. WT blood TFR cells potently suppress antibody production.NP-specific antibody titers from experiments as in FIG. 8A in which WTTFH or WT TFH plus WT TFR cells were transferred into CD28^(−/−)recipients.

FIG. 8C. CD138⁺ plasma cell percentages in draining lymph nodes ofCD28^(−/−) recipients following no transfer (Control), Blood TFHtransfer (Blood TFH) or Blood TFH plus TFR cell transfer (Blood TFH TFR)24 days after immunization. Cells are gated as a percentage ofCD11b⁻CD11c⁻Ly6c⁻ (dump) cells.

FIG. 8D. Quantitation of CD138 plasma cells as gated in FIG. 8C indraining lymph node, spleen and bone marrow.

FIG. 8E. Blood TFH and/or TFR Transfer into TCRα^(−/−) recipients usingexperimental design as in FIG. 8A. Comparison of NP-specific antibodytiters in (1) WT control mice, (2) TCRα^(−/−) recipients given no cells,(3) TCRα^(−/−) recipients given WT blood TFR cells alone, (4) TCRα^(−/−)recipients given total blood CD4 T cells from CXCR5^(−/−) mice immunizedwith NP-OVA 8 days previously, (5) TCRα^(−/−) recipients given bloodCD4⁺FoxP3⁻ cells from unimmunized FoxP3−GFP mice, (6) TCRα^(−/−)recipients given WT blood TFH cells, and (7) TCRα^(−/−) recipients givenWT blood TFH cells plus TFR cells. NP specific IgG levels weredetermined by ELISA.

FIG. 8F. CD138⁺ plasma cells from the spleen (gated as a percent of livecells) were quantified from experiments in FIG. 8E 12 days aftersecondary immunization. Error bars indicate standard error of at leastthree separate experiments.

FIG. 8G. CD4⁺FoxP3⁻ TFH cells from the draining lymph node pre-gated onCD4⁺FoxP3⁻ were quantified from experiments in FIG. 8E 12 days aftersecondary immunization. Error bars indicate standard error of at leastthree separate experiments.

FIG. 8H. Blood TFH cells can have an enhanced ability to stimulateantigen-specific antibody production compared to lymph node TFH cells.Blood TFH cells and draining lymph node TFH cells were isolated from WTmice immunized with NP-OVA 8 days previously and 4×10⁶ cells weretransferred into TCRα^(−/−) mice and immunized as in FIG. 8E.

FIG. 8I. Blood TFR cell suppression is aided by the follicular program.Blood TFH cells were transferred to TCRα^(−/−) mice along with bloodCXCR5− FoxP3 GFP⁺ cells from FoxP3 reporter mice or blood TFR cells.Antibody titers were quantified 12 days after NP-OVA immunization and NPIgG levels are expressed as a percent of TFH transfer group. Dataindicate standard error of at least three independent experiments.

FIG. 8J. PD-1 deficient blood TFR cells more potently suppress antibodyproduction in vivo compared to WT TFR cells. 4×10⁴ WT blood TFH and1.5×10⁴ WT or PD-1 deficient blood TFR cells from mice immunized withNP-OVA 8 days previously were transferred into CD28^(−/−) mice.Recipient mice were immunized with NP-OVA, and NP specific antibodytiters were measured from serum 12 days later. Data are representativeof two independent experiments.

FIG. 8K. PD-1 deficient blood TFR cells more potently suppress antibodyproduction in vivo compared to WT TFR cells. 4×10⁴ WT blood TFH and1.5×10⁴ WT or PD-1 deficient blood TFR cells from mice immunized withNP-OVA 8 days previously were transferred into TCRα^(−/−) mice.Recipient mice were immunized with NP-OVA, and NP specific antibodytiters were measured from serum 12 days later. Data are representativeof two independent experiments.

FIG. 9. Blood Tfh and Tfr cells are present in human blood. Peripheralblood mononuclear cells were isolated from the blood of a healthyindividual by sucrose density centrifugation and stained for indicatedproteins and were analyzed by flow cytometry. Numbers indicatepercentages contained within gates.

FIG. 10A. PD-1 controls TFR cells in NP-OVA immunized mice. WT orPD-1^(−/−) mice were immunized with NP-OVA emulsified in CFA and 7 dayslater draining lymph nodes were stained for CD4⁺FoxP3⁺ICOS⁺CXCR5⁺CD19⁻ Tfollicular regulatory cells (TFR) and CD4⁺FoxP3⁻ICOS⁺CXCR5⁺CD19⁻ Tfollicular helper cells (TFH) using gating strategy shown.

FIG. 10B TFH cells were gated and expressed as a percentage ofCD4⁺FoxP3⁻ cells.

FIG. 10C. TFR cells were gated and expressed as a percentage of all CD4⁺cells.

FIG. 10D. TFR cells were gated and expressed as a percentage ofCD4⁺FoxP3⁺ cells.

FIG. 10E. TFR cells were gated and expressed as a percentage ofCD4⁺CXCR5⁺ICOS⁺ cells.

FIG. 11A. Comparison of PD-L1, CD103 and GITR expression on WT andPD-1^(−/−) T Follicular Regulatory Cells. WT or PD-1^(−/−) mice wereimmunized with MOG (35-55) and 7 days later draining lymph nodes werestained. TFR (CD4⁺ICOS⁺CXCR5⁺FoxP3⁺CD19⁻) cells were analyzed forsurface expression of PD-L1.

FIG. 11B. Comparison of PD-L1, CD103 and GITR expression on WT andPD-1^(−/−) T Follicular Regulatory Cells. WT or PD-1^(−/−) mice wereimmunized with MOG (35-55) and 7 days later draining lymph nodes werestained. TFR (CD4⁺ICOS⁺CXCR5⁺FoxP3⁺CD19⁻) cells were analyzed forsurface expression of CD103.

FIG. 11C. Comparison of PD-L1, CD103 and GITR expression on WT andPD-1^(−/−) T Follicular Regulatory Cells. WT or PD-1^(−/−) mice wereimmunized with MOG (35-55) and 7 days later draining lymph nodes werestained. TFR (CD4⁺ICOS⁺CXCR5⁺FoxP3⁺CD19⁻) cells were analyzed forsurface expression of GITR.

FIG. 12. WT and PD-1 deficient TFR cells express similar levels of GITR.WT and PD-1^(−/−) mice were immunized with NP-OVA and 7 days laterpopulations were sorted according to gating strategy.

FIG. 13A. TFH and TFR cells can bind similar levels of anti-CD3. Gatingof TFH, TFR and B cells from in vitro suppression assays as in FIG. 4K.

FIG. 13B. Cells gated as in FIG. 13A were stained with an anti-hamstersecondary antibody to quantify the amount of anti-CD3 bound to thesurface of the cells.

FIG. 14A. Blood TFH and TFR cells express central memory homing markers.WT and PD-1^(−/−) mice were immunized with MOG/CFA and 7 days laterpopulations from blood were analyzed for CD62L expression.

FIG. 14B. Blood TFH and TFR cells express central memory homing markers.WT and PD-1^(−/−) mice were immunized with MOG/CFA and 7 days laterpopulations from blood were analyzed for CD44 expression.

FIG. 14C. PD-1 expression was compared on WT and PD-1 deficient micefrom draining lymph node and blood TFH and TFR cells 7 days after NP-OVAimmunization.

FIG. 15A. Circulating TFH and TFR cells require dendritic cells fordevelopment. WT or CD11c^(DTR) bone marrow chimeras were immunized withNP-OVA subcutaneously and diphtheria toxin (DT) was administered (ornot) on days 0, 2, 4, and 6 to deplete DCs.

FIG. 15B. Draining lymph node analysis of CD11c⁺MHC II⁺ DCsQuantification of Ki67+TFH (CD4⁺ICOS⁺CXCR5⁺FoxP3⁻Ki67⁺CD19⁻) and TFR(CD4⁺ICOS⁺CXCR5⁺FoxP3⁺Ki67⁺CD19⁻) cells in the draining lymph node andblood. Data are means+/−standard error with 5 mice per group. Data arerepresentative of at least 2 independent experiments.

FIG. 15C. WT or Ciita^(−/−) bone marrow derived dendritic cells (BMDCs)were pulsed with NP-OVA in an overnight in vitro culture, washed andadoptively transferred subcutaneously to WT mice. 5 days later draininglymph nodes and blood were analyzed for ICOS and CXCR5 expression on CD4T cells. WT mice that received no transfer were included as controls.Plots are pregated on CD4⁺CD19⁻. Representative plots are shown.

FIG. 15D. Quantification of draining lymph node and blood TFH (top) andTFR (bottom) cells in BMDC adoptive transfer experiments as in FIG. 15C.Data are means+/−standard error with 5 mice per group. Data arerepresentative of at least 2 independent experiments.

FIG. 15E. mMT mice have normal circulating TFH and TFR cells. WT orIghm^(−/−) “mMT” mice were immunized with NP-OVA subcutaneously and 7days later ICOS⁺CXCR5⁺ CD4 T cells were identified. Plots are pregatedon CD4⁺CD19⁻. Representative plots are shown.

FIG. 15F. Quantification of total lymph node or blood CD4⁺ICOS⁺CXCR5⁺(left; as gated in FIG. 15D), TFH (middle), or TFR (right) cells. Dataare means+/−standard error with 5 mice per group. Data arerepresentative of at least 2 independent experiments.

FIG. 15G. Analysis of the blood:lymph node ratio of TFH and TFR cells inWT and μMT mice immunized as in FIG. 15E. Data are means+/−standarderror with 5 mice per group. Data are representative of at least 2independent experiments.

FIG. 16A. Circulating TFH and TFR cells depend on S1P signals to exitthe lymph node. Analysis of CD69 expression on TFH and TFR cells. Gatingof TFH and TFR cells from the lymph node and blood of WT mice immunizedwith NP-OVA 7 days previously (left). Plots are pregated on CD4⁺CD19⁻.Histograms (right) show CD69 expression on TFH and TFR cells from lymphnode (blue) and blood (red) or on CD4⁺ICOS⁻CXCR5⁻ cells. Data aremeans+/−standard error with 5 mice per group; data are representative ofat least 2 independent experiments.

FIG. 16B. Quantification of CD69 on lymph node and blood TFH and TFRcells as in FIG. 16A. CD4⁺ICOS⁻CXCR5⁻CD19⁻ cells are included ascontrols. Data are means+/−standard error with 5 mice per group; dataare representative of at least 2 independent experiments.

FIG. 16C. Quantification of numbers of TFH cells in the lymph node(left; blue) and blood per ml (right; red) after FTY720 treatment for 5days (on days 2, 4, and 6 post immunization followed by analysis on day7) are shown. Data are means+/−standard error with 5 mice per group;data are representative of at least 2 independent experiments.

FIG. 16D. Quantification of numbers of TFR cells in the lymph node(left; blue) and blood (right; red) after FTY720 treatment for 5 days,as in FIG. 16C. Data are means+/−standard error with 5 mice per group;data are representative of at least 2 independent experiments.

FIG. 16E. Numbers of TFH (left) and TFR (right) cells (per ml blood)after FTY720 treatment during the last 3 or 6 hours of a 7 dayimmunization. Data are means+/−standard error with 5 mice per group;data are representative of at least 2 independent experiments.

FIG. 16F. Representative plots of total CD4⁺ICOS⁺CXCR5⁺ cells from thedraining lymph node (dLN), efferent lymph (lymph) and blood of WT miceimmunized 7 days previously. Plots are pregated on CD4+CD19−. Data arerepresentative from at least 3 replicates.

FIG. 16G. Quantification of percentages of TFH and TFR cells in thedraining lymph node (blue), efferent lymph (green) and blood (red) of WTmice immunized 7 days previously. Data are representative from at least3 replicates.

FIG. 16H. Histograms showing ICOS expression on draining lymph node(blue), lymph (green) and blood (red) TFH (left) and TFR (right) cellsas in FIG. 16G.

FIG. 17A. Circulating TFH and TFR cells migrate to diverse secondarylymphoid organs and tissues. 20, Actin^(CFP)-Fox^(GFP) mice wereimmunized with NP-OVA subcutaneously and 7 days later 2×10⁴ sorted bloodCD4⁺CXCR5⁺CD19⁻ cells were adoptively transferred into CD28 deficientmice which were immunized with NP-OVA. Organs were harvested 7 dayslater for analyses. Transferred cells from indicated organs wereidentified as CFP positive according to gates: iLN; draining inguinallymph node, aLN; axillary lymph node, cLN; cervical lymph node, mLN;mesenteric lymph node, Skin; skin around site of immunization. Plots arepregated on CD4⁺CD19⁻.

FIG. 17B. Dual color overlay plots showing ICOS and CXCR5 expression onCFP positive transferred cells (red) or endogenous CD28 deficient CD4 Tcells (blue) in organs as in FIG. 17A.

FIG. 17C. Quantification of the percentage of TFH cells of theCFP⁺CXCR5⁺ICOS⁺ transferred cells as in FIG. 17B.

FIG. 17D. Quantification of FoxP3+ cell percentages of theCFP⁺CXCR5⁺ICOS⁺ transferred cells.

FIG. 17E. Quantification of the percentage of FoxP3+ cells from thetotal transferred CFP⁺ population gated as in FIG. 17A.

FIG. 17F Interaction of blood TFH cells with IgG1⁺ B cells upon homingto lymph nodes. 2×10⁴ blood TFH (CD4⁺ICOS⁺CXCR5⁺GITR⁻CD19⁻) cells fromNP-OVA immunized CD45.2 mice were adoptively transferred to CD45.1 micethat were then NP-OVA immunized. 7 days later dLN were stained forCD45.2 (Blood TFH), IgG1 or IgD.

FIG. 17G. Interaction of blood TFR cells with IgG1⁺ B cells upon homingto lymph nodes. 1×10⁴ blood TFR (CD4⁺ICOS⁺CXCR5⁺GITR⁺CD19⁻) cells fromNP-OVA immunized CD45.2 mice were adoptively transferred to CD45.1 micethat were then NP-OVA immunized. 7 days later dLNs were stained forCD45.2 (Blood TFR), FoxP3 or IgG1. Data are from transfers of TFH or TFRcells from 20 pooled mice into a single mouse recipient andrepresentative of at least 2 individual experiments.

FIG. 18A. Circulating TFH and TFR cells show enhanced activation uponreentering lymphoid organs of immunized mice in vivo. 20,Actin^(CFP)-Fox^(GFP) mice were immunized with NP-OVA subcutaneously and7 days later 2×10⁴ sorted lymph node or blood CD4⁺CXCR5⁺CD19⁻ cells wereadoptively transferred to CD28 deficient mice that were then immunized.7 days later CFP positive cells in draining lymph nodes were analyzed byflow cytometry. CD28 deficient mice that received no transfer (“NoTrans”) were used as controls.

FIG. 18B. Bcl6 expression in dLN FoxP3⁻CFP⁺ (TFH) cells in CD28^(−/−)recipients of dLN CXCR5 or blood CXCR5 cells as described in FIG. 18A.WT dLN TFH (CD4+ICOS+CXCR5+FoxP3⁻CD19⁻) cells from WT mice immunized 7days previously were used as positive controls and CD28^(−/−) CD4 Tcells from immunized LN were used as negative controls for Bcl6expression, and shown for comparison.

FIG. 18C. ICOS expression on lymph node or blood FoxP3⁻CFP⁺ (TFH) cells(assayed population) which originated from either dLN or blood CXCR5transfers (CXCR5 Transfer) as in FIG. 18A. Total CD4 T cells, dLN andblood TFH from WT immunized mice were included for comparison.

FIG. 18D. ICOS expression on lymph node or blood FoxP3⁺CFP⁺ (TFR) cells(assayed population) which originated from either dLN or blood CXCR5transfers (CXCR5 Transfer) as in FIG. 18C.

FIG. 18E. CXCR5 expression on lymph node or blood FoxP3⁻CFP⁺ (TFH) cellsas in FIG. 18C.

FIG. 18F. CXCR5 expression on lymph node or blood FoxP3⁺CFP⁺ (TFR) cellsas in FIG. 18D.

FIG. 18G. Intracellular cytokine production of IL-21, IL-17A or IL-2 bytransferred FoxP3⁻CFP⁺ (TFH cells) from dLN or blood CXCR5 transfers asin FIG. 18A. Data are from transfers of TFH or TFR cells from 20 pooledmice into a single mouse recipient and representative of at least 2individual experiments.

FIG. 19A. Circulating TFH cells Require Dendritic Cells forRestimulation and Have Memory Properties In vitro activation assay inwhich draining lymph node (dLN) or blood TFH cells (sorted asCD4⁺ICOS⁺CXCR5⁺GITR⁻CD19⁻) were plated with B cells or DCs (isolatedfrom lymph nodes of WT mice immunized 7 days previously) in the presenceof anti-IgM and anti-CD3 for 6 days. Cells were stained for CD4+CD19−and intracellularly for Bcl6 and Ki67. Plots are pregated on CD4⁺CD19⁻.Data indicate means+/−standard error of at least 3 individualexperiments.

FIG. 19B. Quantification of data presented in FIG. 19A. Data indicatemeans+/−standard error of at least 3 individual experiments.

FIG. 19C. Intracellular cytokine staining of samples as in FIG. 19Aexcept samples were stimulated with PMA/Iono for 4 hours in the presenceof golgistop. Data indicate means+/−standard error of at least 3individual experiments.

FIG. 19D. Blood TFH cells require DCs for expansion in vivo. 1.5×10⁴blood CD4+CXCR5+CD19− cells from immunized Actin^(CFP)-Fox^(GFP) micewere adoptively transferred to CD11cDTR bone marrow chimeric mice thatwere immunized with NP-OVA and treated or not with DT on days 0, 3 and5. 7 days after immunization the draining lymph nodes were harvested forflow cytometric analysis of total CFP⁺ cells (left) and expression ofCXCR5 on CFP⁺FoxP3⁻ cells (right). data from transfer of TFH or TFRcells from 20 pooled mice into 3 recipients.

FIG. 19E. Circulating Memory blood CXCR5+ cells dominate the germinalcenter reaction upon homing to dLNs after challenge. Schematic diagramof parabiosis experiments (left). Actin^(CFP) Fox^(GFP) mice wereimmunized subcutaneously with NP-OVA at d0 or d7. At d0, mice weresurgically joined to WT mice and circulatory systems were allowed tofreely exchange for 19 days. Mice were then separated and 6 days laterthe WT “recipient” was immunized with NP-OVA. 7 days later organs wereharvested for presence of CFP+ cells. Comparison of CFP+ cells in thenon-draining lymph node naïve gate or CXCR5 gate in specified organs(right). Data from 3 individual mice/parabiotants.

FIG. 19F. Blood TFH and TFR cells persist for 30 days in vivo. 2×10⁴blood CFP⁺ICOS⁺CXCR5⁺CD19⁻ (TFH+TFR) cells were adoptively transferredto WT mice that were immunized with NP-OVA 30 days later (left). Typicalflow cytometry plots and quantification of data (right).“End”=endogenous CD4 T cells. Adoptive transfer recipients are comparedto mice that did not receive CFP+ cells, “No Trans”. Data from 25 pooledmice transferred to a single mouse recipient and repeated 4 times.

FIG. 20A. TFR cells suppress TFH activation and B cell class switchrecombination in vitro. dLN TFR suppression assays in which dLN TFHCD4+ICOS+CXCR5+GITR−CD19−) and/or TFR (CD4+ICOS+CXCR5+GITR+CD19−) cellswere plated with either B cells or DCs (isolated from the dLN of WT miceimmunized with NP-OVA 7 days previously) for 5-6 days with anti-CD3 andanti-IgM. TFH cells were gated as CD4⁺CD19⁻FoxP3⁻ and stained forsurface expression of CXCR5. Representative histograms (left) andquantification (right) are shown. Data indicate means+/−standard errorof replicate wells and representative of at least 3 independentexperiments.

FIG. 20B. TFH cells gated as in FIG. 20A were intracellularly stainedfor (B) Bcl6 with representative histograms (left) and quantification(right) shown. Data indicate means+/−standard error of replicate wellsand representative of at least 3 independent experiments.

FIG. 20C. TFH cells gated as in FIG. 20A were intracellularly stainedfor (Ki67, with representative histograms (left) and quantification(right) shown. Data indicate means+/−standard error of replicate wellsand representative of at least 3 independent experiments.

FIG. 20D. Intracellular cytokine staining in TFH cells from suppressionassays in which dLN TFH and/or TFR cells were plated with B cells as inFIG. 20A. Data indicate means+/−standard error of replicate wells andrepresentative of at least 3 independent experiments.

FIG. 20E. B cells from suppression assays in which dLN TFH and/or TFRcells were plated with B cells as in FIG. 20A and quantified for surfaceexpression of GL7. Data indicate means+/−standard error of replicatewells and representative of at least 3 independent experiments.

FIG. 20F. Analyses of B cells from suppression assays in which no TFH orTFR cells were added, TFH cells alone were added, TFH and dLNFoxP3+CXCR5− cells from WT unimmunized mice were added, or TFH and TFRcells were added; B cells were stained for GL7 and CD138. Plots arepregated on CD19⁺CD4⁻.

FIG. 20G. Intracellular expression of Ki67 in B cells from suppressionassays in which dLN TFH and/or TFR cells were plated with B cells as inFIG. 20A. Data indicate means+/−standard error of replicate wells andrepresentative of at least 3 independent experiments.

FIG. 20H. Intracellular expression of IgG1 in B cells from suppressionassays in which dLN TFH and/or TFR cells were plated with B cells as inFIG. 20A. Data indicate means+/−standard error of replicate wells andrepresentative of at least 3 independent experiments.

FIG. 20I. Surface staining of GL7 and intracellular staining of IgG1 inB cells from suppression assays in which B cells were cultured with dLNTFH cells alone or with CD4⁺FoxP3⁺CXCR5⁻ cells from LN of unimmunizedFoxP3 GFP reporter mice, or dLN TFR cells. B cells cultured withCD4⁺CXCR5⁻FoxP3⁻CD62L⁺ naïve cells were included as controls. Dataindicate means+/−standard error of replicate wells and representative ofat least 3 independent experiments.

FIG. 21A. Blood TFR cells are less suppressive than LN TFR cells. GL7and IgG1 expression in B cells from suppression assays in which dLN orblood TFR (CD4+ICOS+CXCR5+GITR+CD19−) cells from WT mice immunized 7days previously were added to dLN TFH cells and B cells as in (FIG. 6A)for 6 days. Gating strategy (left) and quantification (right) are shown.Plots are pregated on CD4⁺CD19⁻ FoxP3⁻. Data indicate means+/−standarderror of at least 3 independent experiments.

FIG. 21B. GL7 expression on B cells from suppression assays with dLN TFRcells and blood TFR cells as in FIG. 21A.

FIG. 21C. Intracellular Ki67 staining in TFR cells from suppressionassays in which dLN TFH cells were plated with either B cells or DCsalong with dLN TFR cells or blood TFR cells. Histograms are pregated onCD4+CD19−FoxP3+. Data for individual plots that are representative of atleast two experiments.

FIG. 21D. Blood TFH and TFR cells are capable of eliciting more potent Bcell activation in vivo. Actin^(CFP)-Fox^(GFP) mice were immunized withNP-OVA and 7 days later 2×10⁴ blood or dLN CD4⁺ICOS⁺CXCR5⁺ (TFH and TFR)cells (with the same TFH/TFR ratio) were adoptively transferred to CD28deficient mice that were then immunized with NP-OVA. 7 days laterdraining lymph nodes were harvested and B cells were stained for GL7 andFAS (plots are pregated on CD19) or (E) CD138 and CD19 (pregated on livecells). Data for individual plots that are representative of at leasttwo experiments.

FIG. 21E. Blood TFH and TFR cells are capable of eliciting more potent Bcell activation in vivo. Actin^(CFP)-Fox^(GFP) mice were immunized withNP-OVA and 7 days later 2×10⁴ blood or dLN CD4⁺ICOS⁺CXCR5⁺ (TFH and TFR)cells (with the same TFH/TFR ratio) were adoptively transferred to CD28deficient mice that were then immunized with NP-OVA. 7 days laterdraining lymph nodes were harvested and B cells were stained for CD138and CD19 (pregated on live cells). Data for individual plots that arerepresentative of at least two experiments.

FIG. 22A. TFH and TFR cells from the circulation show decreasedexpression of CXCR5 and ICOS compared to lymph node TFH and TFR cells.WT mice were immunized (or not) with NP-OVA in CFA subcutaneously and 7days later the draining lymph node and blood were collected and analyzedby flow cytometry. Populations of ICOS⁺CXCR5⁺ T follicular helper (TFH)cells in the lymph node (LN; left) and blood (right) of WT mice. Plotsare pregated on CD4⁺FoxP3⁻CD19.

FIG. 22B. Quantification of TFH cells in lymph node (left) and blood(right) from plots as in FIG. 22A. Total CD4+CD19− cells “Total CD4” areincluded as controls.

FIG. 22C Quantification of CXCR5 expression, on lymph node and blood TFHcells gated as in FIG. 22A.

FIG. 22D. ICOS expression on lymph node and blood TFH cells gated as inFIG. 22A.

FIG. 22E. Quantification of Ki67 expression on lymph node and blood TFHcells gated as in FIG. 22A.

FIG. 22F. Populations of ICOS⁺CXCR5⁺ T follicular regulatory (TFR) cellsin the lymph node and blood of WT mice immunized or not with NP-OVA 7days previously. Plots are pregated on CD4⁺FoxP3⁺CD19⁻.

FIG. 22G. Draining LN TFR cells expressed lower levels of CXCR5 than dLNTFH cells.

FIG. 22H. Blood TFR cells expressed even lower levels of CXCR5 than dLNTFR cells.

FIG. 22I. ICOS expression was also greatly attenuated in blood TFR cellscompared to dLN TFR cells.

FIG. 22J. Ki67 intracellular staining revealed that there were similarproportions of dLN and blood TFR cells in cell cycle.

FIG. 23. Comparison of TFR and Treg gene expression signatures (left)Top hits (oriented top to bottom) and (right) bottom hits (orientedbottom to top) of differentially expressed genes in WTCD4+ICOS+CXCR5+GITR+TFR cells (WT) compared to CD4+CXCR5−FoxP3+ non-TFRTregs (Fox) in microarray analysis. PD-1 deficient TFR cells (PD1)(which have increased suppressive capacity) are also shown. Each rowindicates one gene. Data indicate top 100 and bottom 100 hits comparingWT TFR to Tregs. Black indicates row maximum expression, white indicatesrow minimum expression.

FIG. 24. Surface receptors differentially expressed by human blood TFRcells. Top 56 hits from a survey of surface receptor expression on humanblood CD4+CXCR5+ICOS+FoxP3+CD19− TFR, CD4+CXCR5+ICOS+FoxP3−CD19− TFH,CXCR5− CD4 (CD4) and CXCR5− Tregs (Treg) are assessed at the proteinlevel by flow cytometry. Hits are sorted based on receptorsdifferentially expressed by TFR and TFH cells. Id indicates gene name.Value indicates mean fluorescence intensity on TFR cells. Heat mapindicates relative high expression (black) or low expression (white).

FIG. 25. Surface receptors differentially expressed by human blood TFHcells. Top 19 hits from a survey of surface receptor expression on humanblood CD4+CXCR5+ICOS+FoxP3+CD19− TFR, CD4+CXCR5+ICOS+FoxP3−CD19− TFH,CXCR5− CD4 (CD4) and CXCR5− Tregs (Treg) are assessed at the proteinlevel by flow cytometry. Hits are sorted based on receptorsdifferentially expressed by TFH and TFR cells. Id indicates gene name.Fold change indicates MFI fold increase on TFH versus TFR cells. Valueindicates mean fluorescence intensity on TFR cells. Heat map indicatesrelative high expression (black) or low expression (white).

FIG. 26. Blockade of the PD-1 pathway can heighten antibody stimulatingcapacity of TFH cells. In vitro class switch recombination assay inwhich murine B cells are plated with sorted murine TFH cells to induceIgG1 class switch recombination. In some wells an anti-PDL1 blockingantibody was added to inhibit the PD-1 pathway.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

So that the invention may be more readily understood, certain terms arefirst defined.

T follicular regulatory (TFR) cells as used herein include, but are notlimited to, the following cell surface markers:CD4+ICOS+CXCR5+FoxP3+CD19−. or CD4+ICOS+CXCR5+GITR+CD19−, orCD4+ICOS+CXCR5+CD25hiCD19−. In one embodiment, TFR cells have thefollowing cell surface markers: CD4⁺CXCR5⁺ICOS⁺ and at least one surfacemarker selected from: GITR⁺, CD25^(hi), CD162, CD27, CD95, CD9, CD43,CD50, CD45RB, CD102, CD61, CD58, CD196, CD38, CD31, CD15, CD25, CD13,CD66a/c/e, CD1b CD63, CD32, CD97, HLA-HQ, CD150, Siglec-9, Integrinβ7,CD71, CD180, CD218a, CD193, CD235ab, CD35, CD140a, CD158b, CD33, CD210,HLA-G, CD167a, CD119, CX3CR1, CD146, HLA-DR, CD85, CD172b, SSEA-1,CD49c, CD170, CD66b, and CD86. In one embodiment, TFR cells have thefollowing cell surface markers: CD4⁺CXCR5⁺ICOS⁺ and at least one surfacemarker selected from: CD27, CD278 (ICOS), CD150, Siglec-9, CD140a,CD158b, CD33.

T follicular helper (TFH) cells as used herein include, but are notlimited to the following cell surface markers:CD4+ICOS+CXCR5+FoxP3−CD19−. In one embodiment, TFH cells have thefollowing cell surface markers: CD4, CXCR5, ICOS positive and at leastone marker selected from CD163, CD127, CD8a, CD89, CD197, CD161, CD6,CD229, CD96, CD272, CD148, CD107a, CD100, CD82, CD126, CD45RO, CD279,CD5, and CD99 and optionally wherein the TFH cells are negative for oneor more of the following receptors GITR, CD25, CD162, CD27, CD95, CD9,CD43, CD50, CD45RB, CD102, CD61, CD58, CD196, CD38, CD31, CD15, CD25,CD13, CD66a/c/e, CD11b CD63, CD32, CD97, HLA-HQ, CD150, Siglec-9,Integrinβ7, CD71, CD180, CD218a, CD193, CD235ab, CD35, CD140a, CD158b,CD33, CD210, HLA-G, CD167a, CD119, CX3CR1, CD146, HLA-DR, CD85, CD172b,SSEA-1, CD49c, CD170, CD66b, and CD86. In one embodiment, TFH cells havethe following cell surface markers: CD4, CXCR5, ICOS positive and atleast one marker selected from CD163, CD127, CD161, CD6, CD229, CD272,CD100, CD126, PD-1 (CD279), and optionally wherein the TFH cells arenegative for one or more of the following receptors GITR, CD25, CD162,CD27, CD95, CD9, CD43, CD50, CD45RB, CD102, CD61, CD58, CD196, CD38,CD31, CD15, CD25, CD13, CD66a/c/e, CD11b CD63, CD32, CD97, HLA-HQ,CD150, Siglec-9, Integrinβ7, CD71, CD180, CD218a, CD193, CD235ab, CD35,CD140a, CD158b, CD33, CD210, HLA-G, CD167a, CD119, CX3CR1, CD146,HLA-DR, CD85, CD172b, SSEA-1, CD49c, CD170, CD66b, and CD86.

In one embodiment, TFH cells have the following cell surface markers:CD4, CXCR5, ICOS positive and at least one marker selected from CD163,CD127, CD161, CD6, CD229, CD272, CD100, CD126, PD-1 (CD279), andoptionally wherein the following markers are expressed a lower levels onTFH cells as compared to the levels of expression on TFR cells whereinsuch receptors are selected from: GITR, CD25, CD162, CD27, CD95, CD9,CD43, CD50, CD45RB, CD102, CD61, CD58, CD196, CD38, CD31, CD15, CD25,CD13, CD66a/c/e, CD11b CD63, CD32, CD97, HLA-HQ, CD150, Siglec-9,Integrinβ7, CD71, CD180, CD218a, CD193, CD235ab, CD35, CD140a, CD158b,CD33, CD210, HLA-G, CD167a, CD119, CX3CR1, CD146, HLA-DR, CD85, CD172b,SSEA-1, CD49c, CD170, CD66b, and CD8.

T regulatory cells (Tregs) as used herein include, but are not limitedto the following cell surface markers: CD4+ GITR+ CXCR5⁻ or CD4+FoxP3+CXCR5⁻ or CD4+ CD25hi CXCR5⁻.

Populations of TFH or TFR cells referred to herein as “isolated” orpurified” from blood refers to cells that have been removed from thebody as part of a sample taken from the peripheral blood, organs ortissues of a subject. “Isolated” and “purified” cell compositions mayfurther be enriched for the desired cell type via known procedures forseparating desired cell types from other cell populations in a sampleincluding cell sorting. As used herein “enriched” means that theresulting sample comprises more of the desired cell type than other celltypes in the sample.

The terms “inhibit”, “inhibition, “suppress” and “suppression” in termsof an immune response includes the decrease, limitation or blockage of,for example a particular action, function or interaction (e.g. antibodysuppression).

The terms “enhance”, promote” or “stimulate” in terms of an immuneresponse includes an increase, facilitation, proliferation, for examplea particular action, function or interaction associated with an immuneresponse (e.g. increase in antibody production).

As used herein, the term “modulate” includes up-regulation anddown-regulation, e.g., enhancing or inhibiting an immune response. Theterm “modulate” when used with regard to modulation of a receptorincludes up-regulation or down-regulation of the biological activityassociated with that receptor when the receptor is activated, forexample, by its ligand or inhibited, for example, with a blockingantibody.

As used herein, the term “immune cell” refers to cells that play a rolein the immune response. Immune cells are of hematopoietic origin, andinclude lymphocytes, such as B cells and T cells; natural killer cells;myeloid cells, such as monocytes, macrophages, eosinophils, mast cells,basophils, and granulocytes.

As used herein, the term “T cell” includes CD4+ T cells and CD8+ Tcells. The term “antigen presenting cell” includes professional antigenpresenting cells (e.g., B lymphocytes, monocytes, dendritic cells,Langerhans cells) as well as other antigen presenting cells (e.g.,keratinocytes, endothelial cells, astrocytes, fibroblasts,oligodendrocytes).

The term “native” cells or “wild-type” cells as used herein withreference to, for example, TFR cells, TFH cells or other cells, meansthat the cells are essentially phenotypically and functionally the sameas those cells of the same cell-type generally found at the originalsource of the native or wild type cells, such as, for example, TFR cellsnormally found in the blood, organs or tissue of a subject.

The term TFR cells with “enhanced suppressive capacity” or “enhancedimmune suppressive activity” or “enhanced regulatory capacity” refers toTFR cells that have been activated in the presence of a PD-1 or PD-1ligand antagonist such that they have enhanced immune suppressiveactivity as compared to native TFR cells. Enhanced immune suppressiveactivity may be measured by standard in vivo and in vitro assays such asantibody suppression assays as are known in the art and describedherein.

The term TFH cells with “enhanced stimulatory capacity”, “enhancedimmune stimulatory capacity” or “enhanced antibody stimulatory capacity”refers to TFH cells that have been activated in the presence of a PD-1or PD-1 ligand antagonist such that they have enhanced stimulatorycapacity as compared to native TFR cells. Enhanced stimulatory capacitymay be measured, for example, by the novel in vivo and in vitro antibodyproliferation assays of the invention as described herein.

As used herein, the term “immune response” includes T cell mediatedand/or B cell mediated immune responses and include those immuneresponses that are mediated by TFR cells or TFH cells. Exemplary immuneresponses include T cell responses, e.g., cytokine production, andcellular cytotoxicity. In addition, the term immune response includesimmune responses that are indirectly affected by T cell activation,e.g., antibody production (humoral responses) and activation of cytokineresponsive cells, e.g., macrophages.

A “subject” is preferably a human subject but can also be any mammal,including an animal model; in which modulation of an autoimmune reactionis desired. Mammals of interest include, but are not limited to:rodents, e.g. mice, rats; livestock, e.g. pigs, horses, cows, etc.,pets, e.g. dogs, cats; and primates. A subject may also be a donor ofperipheral blood T cells who is not the subject in which modulation ofan autoimmune reaction is desired also referred to herein as a “healthydonor”. A subject may also be referred to herein as a “patient”.

The terms “treatment” “treat” and “treating” encompasses alleviation,cure or prevention of at least one symptom or other aspect of adisorder, disease, illness or other condition (collectively referred toherein as a “condition”), or reduction of severity of the condition, andthe like. A composition of the invention need not affect a completecure, or eradicate every symptom or manifestation of a disease, toconstitute a viable therapeutic agent. As is recognized in the pertinentfield, drugs employed as therapeutic agents may reduce the severity of agiven disease state, but need not abolish every manifestation of thedisease to be regarded as useful therapeutic agents. Beneficial ordesired clinical results include, but are not limited to, alleviation ofsymptoms, diminishment of extent of disease, stabilization (i.e., notworsening) of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, remission (whetherpartial or total, whether detectable or undetectable) and prevention ofrelapse or recurrence of disease. Similarly, a prophylacticallyadministered treatment need not be completely effective in preventingthe onset of a condition in order to constitute a viable prophylacticagent. Simply reducing the impact of a disease (for example, by reducingthe number or severity of its symptoms, or by increasing theeffectiveness of another treatment, or by producing another beneficialeffect), or reducing the likelihood that the disease will occur orworsen in a subject, is sufficient.

“Treatment” can also mean prolonging survival as compared to expectedsurvival if not receiving treatment. In one embodiment, an indicationthat a therapeutically effective amount of a composition has beenadministered to the patient is a sustained improvement over baseline ofan indicator that reflects the severity of the particular disorder.

By a “therapeutically effective amount” of a composition of theinvention is meant an amount of the composition which confers atherapeutic effect on the treated subject, at a reasonable benefit/riskratio applicable to any medical treatment. The therapeutic effect issufficient to “treat” the patient as that term is used herein.

As used herein, “cell therapy” is a method of treatment involving theadministration of live cells.

“Adoptive immunotherapy” is a treatment process involving removal ofcells from a subject, the processing of the cells in some manner ex-vivoand the infusion of the processed cells into the same subject as atherapy.

As used herein, a vaccine is a composition that provides protectionagainst a viral infection, cancer or other disorder or treatment for aviral infection, cancer or other disorder. Protection against a viralinfection, cancer or other disorder will either completely preventinfection or the tumor or other disorder or will reduce the severity orduration of infection, tumor or other disorder if subsequently infectedor afflicted with the disorder. Treatment will cause an amelioration inone or more symptoms or a decrease in severity or duration. For purposesherein, a vaccine results from co-infusion (either sequentially orsimultaneously) of an antigen and a composition of cells produced by themethods herein. As used herein, amelioration of the symptoms of aparticular disorder by administration of a particular composition refersto any lessening, whether permanent or temporary, lasting or transientthat can be attributed to or associated with administration of thecomposition.

As used herein a “vaccination regimen” means a treatment regimen whereina vaccine comprising an antigen and/or adjuvant is administered to asubject in combination with for example, composition of the inventioncomprising TFR cells and/or TFH cells, simultaneously, in eitherseparate or combined formulations, or sequentially at different timesseparated by minutes, hours or days, but in some way act together toprovide the desired enhanced immune response to the vaccine in thesubject as compared to the subject's immune response in the absence of aTFR and/or TFH composition in accordance with the invention.

The term “adjuvant” is used in its broadest sense as any substance whichenhances, increases, upwardly modulates or otherwise facilitates animmune response to an antigen. The immune response may be measured byany convenient means such as antibody titre or level of cell-mediatedresponse.

“Immune-related disease” means a disease in which the immune system isinvolved in the pathogenesis of the disease. Subsets of immune-relateddiseases are autoimmune diseases. Autoimmune diseases include, but arenot limited to, rheumatoid arthritis, myasthenia gravis, multiplesclerosis, psoriasis, systemic lupus erythematosus, autoimmunethyroiditis (Hashimoto's thyroiditis), Graves' disease, inflammatorybowel disease, autoimmune uveoretinitis, polymyositis, and certain typesof diabetes. Other immune-related diseases are provided infra. In viewof the present disclosure, one skilled in the art can readily perceiveother autoimmune diseases treatable by the compositions and methods ofthe present invention.

A disease or condition wherein modulation of, and preferably selectivemodulation of, TFR cells and/or TFH cells is therapeutic, includesdiseases wherein suppression of a pathogenic antibody response isdesired and diseases where enhancement of a protective antibody responseis desired. In some instances, for example, in a disease in whichsuppression of a pathogenic antibody response is therapeutic, it iscontemplated herein that the disease may be treated by selectivelyup-regulating TFR cell-mediated antibody suppression whilesimultaneously selectively down-regulating TFH cell-mediated immuneresponse.

Examples of diseases or conditions wherein suppression of a pathologicalantibody response is desired include diseases in which antibodiescontribute to, or are primarily responsible for pathogenesis. Suchdiseases or conditions in which antibodies contribute to and/or areprimarily responsible for pathogenesis include, but are not limited to,diabetes (Type 1), multiple sclerosis, systemic lupus erythematosus,allergy, asthma, multiple sclerosis, myasthenia gravis, lupuserythematosus, autoimmune hemolytic, scleroderma and systemic sclerosis,Sjogren's syndrom, undifferentiated connective tissue syndrome,antiphospholipid syndrome, vasculitis (polyarteritis nodosa, allergicgranulomatosis and angiitis, Wegner's granulomatosis, hypersensitivityvasculitis, polymyositis systemic lupus erythematosus, collagendiseases, autoimmune hepatitis, primary (autoimmune) sclerosingcholangitis or other hepatic diseases, thyroiditis, glomerulonephritis,Devic's disease, autoimmune throbocytopenic purpura, pemphigus vulgaris,vasculitis caused by ANCA, Goodpasture's syndrome, rheumatic fever,Grave's disease (hyperthyroidism), insulin resistant diabetes,pernicious anemia, celiac disease, hemolytic disease of the newborn,cold aggutinin disease, IgA nephropathology, glomerulonephritis(including post-streptococcal), primary biliary cirhosis, and serumsickness. In one embodiment diseases in which pathogenic antibodiescontribute to and/or are primarily responsible for pathogenesis areselected from multiple sclerosis, systemic lupus erythematosis, allergy,myasthenia gravis, collagen diseases, glomerulonephritis, Devic'sdisease, vasculitis caused by ANCA, and celiac disease.

Examples of diseases or conditions wherein enhancement of a protectiveantibody response is desired includes those diseases in which thepresence of a robust antibody response reduces or eliminates the causesor pathogenesis of the disease. Examples of diseases or conditionsbenefiting from a protective antibody response include, but are notlimited to viral infections and cancer.

While not a disease or condition, enhancement of a protective antibodyresponse is also beneficial in a vaccine or as part of a vaccinationregimen as is described herein.

An agent that is an “antagonist” of a cell surface receptor on a TFHcell or a TFR cell is an agent which down regulates or blocks thebiological function of the cell surface receptor. As used herein, anagent which is an “antagonist” includes agents that bind or otherwiseinterfere with ligands of cell surface receptor thereby blocking theability of the ligand to bind to the cell surface receptor anddown-regulate or prevent the biological function of the cell surfacereceptor.

An agent that is an “agonist” of a cell surface receptor on a TFH cellor a TFR cell is an agent which upregulates or increases the biologicalfunction of the cell surface receptor.

II. Starting Population of Cells

In one embodiment TFR cells and TFR precursor cells, for example, Tregulatory (Treg) progenitor cells are derived from a mixed cellpopulation containing such cells (e.g. from peripheral blood, tissue ororgans). Preferably the mixed cell population containing TFR cells orTFR cell precursors is enriched such that TFR cells or TFR cellprecursors comprise more TFR cells than other cell types in thepopulation. In one embodiment, an enriched composition of TFR cells is acomposition wherein the TFR cells make up greater than about 50% (e.g.,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.9% ormore) of the cell population in the composition. In some embodiments,the TFR cells comprise about 90%, 95%, 98%, 99%, 99.5%, 99.9% or more ofthe cells in the composition, and such compositions are referred toherein as “highly purified” or “substantially homogenous” TFR cellcompositions.

While a starting population of TFR cells is described above, it isunderstood that similar procedures may be applied to obtaining astarting population of TFH cells. Accordingly in some embodiments amixed cell population containing TFH is enriched such that thecomposition comprises more TFH cells than other cell types in thepopulation. In one embodiment, an enriched composition of TFH cells is acomposition wherein the TFH cells make up greater than about 50% (e.g.,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.9% ormore) of the cell population in the composition. In some embodiments,the TFH cells comprise about 90%, 95%, 98%, 99%, 99.5%, 99.9% or more ofthe cells in the composition, and such compositions are referred toherein as “highly purified” or “substantially homogenous” TFH cellcompositions. In some embodiments, TFR cells or TFH cells are enrichedfrom a population of cells prior to an activating and/or expanding step.In some embodiments TFR cells or TFH cells are enriched from apopulation of cells after the activating and/or expanding step.

Such highly purified or substantially homogenous populations of TFRcells or TFH cells may be used for in-vivo and in-vitro diagnoses andexamination of TFR cell-mediated, or TFH cell-mediated diseases.

TFR cells can be enriched by targeting for selection of cell surfacemarkers specific for immune suppressive TFR cells and separating usingautomated cell sorting such as fluorescence-activated cell sorting(FACS), solid-phase magnetic beads, etc. To enhance enrichment, positiveselection may be combined with negative selection against cellscomprising surface makers specific to non-T-regulatory cell types, suchas depletion of CD8, CD11b, CD16, CD19, CD36 and CD56-bearing cells.

In one embodiment TFR cells are sorted via flow cytometry based onsurface markers of CD4⁺CXCR5⁺ICOS⁺GITR⁺, or CD4⁺CXCR5⁺ICOS⁺CD25⁺. In oneembodiment, TFR cells are sorted via flow cytometry based on thefollowing cell surface markers: CD4⁺CXCR5⁺ICOS⁺ and at least one surfacemarker selected from one or more of: GITR⁺, CD25^(hi), CD162, CD27,CD95, CD9, CD43, CD278, CD50, CD45RB, CD102, CD61, CD58, CD196, CD38,CD31, CD15, CD25, CD13, CD66a/c/e, CD11b CD63, CD32, CD97, HLA-HQ,CD150, Siglec-9, Integrinβ7, CD71, CD180, CD218a, CD193, CD235ab, CD35,CD140a, CD158b, CD33, CD210, HLA-G, CD167a, CD119, CX3CR1, CD146,HLA-DR, CD85, CD172b, SSEA-1, CD49c, CD170, CD66b, and CD86.

TFH cells may be sorted based on surface markers of CD4, CXCR5, ICOSpositive; GITR negative, CD25 negative. In one embodiment, TFH cells aresorted via flow cytometry based on the following cell surface markers:CD4, CXCR5, ICOS positive and at least one marker selected from: CD163,CD127, CD8a, CD89, CD197, CD161, CD6, CD229, CD96, CD272, CD148, CD107a,CD100, CD82, CD126, CD45RO, CD279, CD5, and CD99, and optionally whereinthe TFH cells are negative for one or more of the following receptorsGITR, CD25, CD162, CD27, CD95, CD9, CD43, CD50, CD45RB, CD102, CD61,CD58, CD196, CD38, CD31, CD15, CD25, CD13, CD66a/c/e, CD11b CD63, CD32,CD97, HLA-HQ, CD150, Siglec-9, Integrin?7, CD71, CD180, CD218a, CD193,CD235ab, CD35, CD140a, CD158b, CD33, CD210, HLA-G, CD167a, CD119,CX3CR1, CD146, HLA-DR, CD85, CD172b, SSEA-1, CD49c, CD170, CD66b, andCD86.

It is believed that these sorting methodologies also contribute to theenhanced suppressive capacity of TFH cells and TFR cells alone or incombination with activating such cells in the presence of an antagonistof PD-1 or PD-1 ligand as is described herein.

In one embodiment, an initial population of TFR cells may also beisolated from the peripheral blood of a subject and further enriched forTFR cells. In one embodiment, an initial population of TFH cells mayalso be isolated from the peripheral blood of a subject and furtherenriched for TFH cells. Methods of purifying TFR cells or TFH cells fromother PBMCs in the blood, using methods such as differentialsedimentation through an appropriate medium, e.g. Ficoll-Hypaque[Pharmacia Biotech, Uppsala, Sweden], and/or methods of cell sorting,are well known and examples of such methods are described herein.

In one embodiment the invention provides a composition of TFR cellsderived from the peripheral blood of a subject (also referred to hereinas “blood TFR cells”) wherein the composition comprises about 90%, 95%,98%, 99%, 99.5%, 99.9% or more of the cells in the composition.

In one embodiment the invention provides a composition of TFH cellsderived from the peripheral blood of a patient (also referred to hereinas “blood TFH cells”) wherein the composition comprises about 90%, 95%,98%, 99%, 99.5%, 99.9% or more of the cells in the composition. Suchhigh purity compositions of blood TFR cells or blood TFH cells may beused directly in methods of modulating the immune system as describedherein. Alternatively such compositions may be further activated and/orexpanded prior to use in modulating the immune system as describedherein.

III. Activation/Expansion of Starting Population of Cells

In one embodiment, the activation of a starting cell population isachieved by contacting the starting population of TFH cells or TFR cellswith T cell stimulatory composition and/or in the presence of a PD-1 orPD-1L antagonist. The activating step may further include an expandingstep or the cell population may be expanded separately from theactivating step. If an expanding step is desired, the cells arepreferably expanded at least 50-fold, and preferably at least 100, 200,300, 500 and 800-fold.

Preferred stimulatory compositions stimulate the T cells by binding andactivating the T cell receptor complex on the cells. In one embodiment,stimulatory compositions may include agents capable of binding andactivating selective TFR and selective TFH receptors as describedherein. In one embodiment the stimulatory compositions comprisephysiological antigen presenting cells (APCs), such as CD19+ B cells(preferably autologous from blood) a TCR/CD3 activator such as amultivalent antibody or ligand for TCR/CD3; a TCR costimulator activatorsuch as multivalent antibody or ligand for CD28, GITR, CD5, ICOS, OX40or CD40L; and optionally an interleukin such as IL-2. In one embodiment,the TCR/CD3 activator is an anti-CD3 antibody, and the TCR costimulatoractivator is an anti-CD28 antibody. The anti-CD3 and anti-CD28antibodies are optionally immobilized on beads as are known in the artand provided in a cell:bead ratio of between 1:1 and 1:2.

In certain embodiments, the stimulatory composition may further includeone or more additional agents, e.g., a costimulatory agent, a secondregulatory T cell stimulatory agent, or agents that generally promotethe survival and/or growth of T cells.

In certain embodiments, the costimulatory agent is an antibody or ligandspecific for a T cell costimulator, such as CD28 or ICOS, as describedbelow. In particular embodiments, the costimulatory agent is an agonistantibody, such as an agonist antibody which binds to CD28.

The stimulatory composition alternatively comprises a second regulatoryT cell stimulatory agent. Exemplary stimulatory agents includegranulocyte colony stimulating factor, interleukins such as IL-2, IL-6,IL-7, IL-13, and IL-15, and hepatocyte growth factor (HGF).

In particular embodiments, one or more components of the stimulatorycomposition is immobilized on a substrate, such as a cell or bead. Cellssuitable for use as substrates include artificial antigen-presentingcells (AAPCs) (Kim, J V et al, Nat Biotechnol. April 2004; 22(4):403-10;and Thomas, A K et al, Clin Immunol. December 2002; 105(3):259-72).Beads can be plastic, glass, or any other suitable material, typicallyin the 1-20 micron range.

Examples of PD-1 and PD-1 ligand antagonists are disclosed in U.S. Pat.No. 7,722,868, incorporated herein by reference. Suitable PD-1 and PD-1ligand antagonists include a PD1-ligand antibody, an anti-PD-1 antibody,a peptide or a small molecule wherein the agent inhibits the interactionbetween PD-1 and a PD-1 ligand. Other examples of PD-1 or PD-1 ligandantagonists are disclosed, for example in U.S. Pat. Nos. 8,168,757;8,114,845; 8,008,449; 7,595,048; 7,488,802; and 7,029,674.

Optimal concentrations of each component of the stimulatorycompositions, culture conditions and duration can be determinedempirically using routine experimentation.

Populations of TFR cells expanded/activated in the presence of a PD-1 orPD-1 ligand antagonist have enhanced suppressive activity asdemonstrated by their ability to inhibit TFH-mediated antibodyproduction in vitro and in vivo as demonstrated in appropriate assays asare described herein.

Populations TFH cells expanded/activated in the presence of a PD-1 orPD-1 ligand antagonist have enhanced antibody stimulatory activity asdemonstrated by their ability to enhance TFH-mediated antibodyproduction in vitro and in vivo as demonstrated in appropriate assaysdescribed herein. Such TFH cells are referred to herein as “TFH cellshaving enhanced stimulatory capacity”.

IV. Modulation of the Immune System

The expanded and/or activated TFR cells and compositions thereof asdescribed herein may be introduced into the subject to treat immunerelated diseases, for example, by modulating an autoimmune reaction. Forexample, the subject may be afflicted with a disease or disordercharacterized by having an ongoing or recurring autoimmune reaction,such as the diseases/disorders including, but not limited to, lupuserythematosus; myasthenia gravis; autoimmune hepatitis; rheumatoidarthritis; multiple sclerosis; Grave's disease, and graft versus hostdisease (GVHD).

In one embodiment, if upregulation of an immune response in a subject isdesired, such as for example, increasing antibody proliferation inresponse to a vaccine, an enriched and optionally expanded and/oractivated composition comprising a starting population of TFH cells maybe administered to a subject.

In one embodiment modulation of the immune system is achieved when uponadministration of a composition of the invention, the ratio of TFR cellsto TFH cells in a subject is changed as compared to the ratio of TFRcells to TFH cells in a subject prior to administration of a compositionof the invention. The ratios of TFR cells to TFH cells in a subjectprior to, and after, administration of a composition of the inventionmay be measured by assaying for the presence of TFR cells or TFH cellsin a patient's blood. Examples of suitable assays for measuring theratio of TFR cells to TFH cells in a patient's blood are describedherein.

In one embodiment, if downregulation of a subject's immune response isdesired, such as, for example, when the subject has an autoimmunedisease and inhibition of an antibody response is desired, a highlypurified composition of blood derived TFR cells, or a compositionenriched for TFR cells having enhanced suppressive activity may beadministered to a patient. After administration of the composition, ablood sample from the patient may be tested to determine if the ratio ofTFR cells to TFH cells is high.

In one embodiment, if upregulation of an immune response in a subject isdesired, such as for example, increasing antibody proliferation inresponse to a vaccine, a highly purified composition of blood-derivedTFH cells may be administered to a patient. After administration of TFHcomposition, a blood sample from the patient may be tested to determineif the ratio of TFH cells to TFR cells is high.

Accordingly, the invention provides methods and compositions foradoptive cellular immunotherapy comprising introducing into a patient inneed thereof an effective amount of the subject's ex vivoexpanded/activated TFR cells, for example. These applications generallyinvolve reintroducing expanded/activated TFR cells extracted from thesame patient, though the methods are also applicable to adoptivecellular immunotherapy for treatment of graft-versus-host diseaseassociated with transplantation, particularly bone marrowtransplantation using TFRs derived from donor tissue, and/or healthyindividuals.

In an exemplary adoptive cell transfer protocol comprises a mixedpopulation of cells is initially extracted from a target donor.Depending on the application, the cells may be extracted during a periodof remission, or during active disease. Typically this is done bywithdrawing whole blood and harvesting PMBCs by, for example,leukapheresis (leukopheresis). For example, large volume leukapherisis(LVL) has been shown to maximize blood leukocyte yield. Harvests reach20×10⁶ cells/L using a continuous flow apheresis device (Spectra, COBEBCT). Symptoms of hypocalcemia are avoided by a continuous infusion ofcalcium administrated throughout leukapheresis. Typically 15-45 litersof fluid corresponding to about 4 total blood volumes are harvestedduring a period of time ranging from about 100 to 300 minutes.

The harvested PMBCs may be separated by flow cytometry or other cellseparation techniques based on Treg and/or TFR-specific cell markerssuch as CD4, CD25, CXCR5, ICOS, and GITR and expanded/activated asdescribed herein, and then transfused to a patient, preferably by theintravenous route, typically the cell donor (except in GVHD where thedonor and recipient are different), for adoptive immune suppression.Alternatively, the cells may be frozen for storage and/or transportprior to and/or subsequent to expansion.

Effective and optimized dosages and treatment regimens using theexpanded and/or enriched and optionally highly pure TFH or TFR cells areknown in the art based on previous clinical experience with existingT-cell infusion therapies, and can be further determined empirically.

The preferred route of administration of the TFR and TFH cellcompositions to a subject in accordance with the invention is by theintravenous route. However, depending on the application, cellcompositions in accordance with the invention may be administered byother routes including, but not limited to, parenteral, oral or byinhalation.

V. Vaccination

The present invention also contemplates a method for enhancing an immuneresponse to an antigen comprising the administration to a subject aspart of a vaccination regimen, TFR cells having enhanced suppressiveactivity, TFR cell compositions derived from peripheral blood and/or TFHcells derived from peripheral blood. The present invention isparticularly useful in pharmaceutical vaccines and genetic vaccines inhumans.

Adjuvants promote the immune response in a number of ways such as tomodify the activities of immune cells that are involved with generatingand maintaining the immune response. Additionally, adjuvants modify thepresentation of antigen to the immune system. The compositions of theinvention may be used as adjuvants in a vaccination regimen.

In one embodiment, compositions of TFR cells in accordance with theinvention may be used in a vaccination regimen. Without being limited toa specific theory, it is believed that TFR cells may control germinalcenter (GC) B cell differentiation into long-lived plasma cells versusmemory B cells thereby enhancing the immune response to the antigen.

In one embodiment compositions of TFH cells, particularly TFH cellsderived from the peripheral blood of a patient (also referred to hereinas “blood TFH cells”) may be used in a vaccination regimen to enhanceTFH cell mediated antibody responses. Without being limited to anyparticular theory, it is believed that TFH cells derived from the bloodmigrate to lymph nodes and interact with cognate B cells rapidly uponantigen exposure, wherein naïve T cells need at least two to four daysto differentiate and upregulate CXCR5. Accordingly, TFH cells derivedfrom blood have greater antibody stimulatory capacity.

In one embodiment, it may be desirable to upregulate an immune responseor downregulate an immune response as part of a vaccination regimen.This can be accomplished by administering compositions enriched for orhighly purified for TFR cells or compositions enriched for or highlypurified TFH cells to change the ratio of TFR cells to TFH cells in asubject's blood in combination with the administration of a vaccine.

VI. Novel In-Vivo and In Vitro Assays

The invention also provides in vivo and in vitro assays to analyze thefunctions of the compositions of TFR cells and TFH cells in accordancewith the invention.

In one exemplary embodiment the invention provides an assay to analyzethe capacity of TFR cells to inhibit activation of naïve CD4 T cells.Briefly, WT and PD-1^(−/−) mice are immunized with MOG/CFA and TFR cellsare sorted from draining lymph nodes and plated 1:1:1 with CF SE-labeledCD4 naïve WT (CD4⁺CD62L⁺FoxP3⁻) responder cells and WT GL7⁻B220⁺ B cellsfrom MOG/CFA immunized mice along with anti-CD3 and anti-IgM for 4 days.3 days later samples are analyzed by flow cytometry. It is understoodthat any suitable antigen/adjuvant combinations may be used to immunizemice and that the cells may be stimulated by any suitable combinationsof stimulatory factors for this assay.

In one exemplary embodiment the invention provides an assay to analyzecapacity of TFR cells to inhibit activation of naïve CD4 T cells. WT andPD-1^(−/−) mice are immunized with MOG/CFA and TFR cells and sorted fromdraining lymph nodes and plated 1:1:1 with CF SE-labeled CD4 naïve WT(CD4⁺CD62L⁺FoxP3⁻) responder cells and WT GL7⁻ B220⁺ B cells fromMOG/CFA immunized mice along with anti-CD3 and anti-IgM for 4 days. 3days later samples are analyzed by flow cytometry. T responders areanalyzed for CD69 expression and proliferation by measuring CFSEdilution.

In one embodiment the invention provides an assay for an in vitro IgGsuppression. Briefly, TFR cells are sorted as in the assay to analyzecapacity of TFR cells to inhibit activation of naïve CD4 T cells and areplated in a 1:1:1 ratio of TFR (CD4⁺ICOS⁺CXCR5⁺GITR⁺CD19⁻), TFH(CD4⁺ICOS⁺CXCR5⁺GITR⁻CD19⁻), and B (GL-7⁻B220⁺) cells from draininglymph nodes of MOG/CFA immunized mice in the presence of anti-CD3 andanti-IgM for 6 days. Total IgG was measured by ELISA from supernatants.In one embodiment the in-vitro suppression assay may be performed over arange of concentrations of anti-CD3. Naive (CD4⁺ICOS⁻CXCR5⁻CD19⁻) cellsfrom immunized mice may be included as controls It is understood thatany suitable antigen/adjuvant combinations may be used to immunize miceand that the cells may be stimulated by any suitable combinations ofstimulatory factors for this assay.

Novel assays of the invention are useful as a diagnostic tool formeasuring a subject's TFR cell function and TFH cell function. Suchassays are useful in the identification and typing of autoimmunediseases.

The assays of the invention are also useful in measuring the ratio ofTFR cells to TFH cells in a patient's blood prior to or during an immuneresponse and/or prior to and after administration of a composition ofthe invention. Such assays are useful as a diagnostic to assist indetermining whether an immune response in a subject should beupregulated or down-regulated or whether an immune modulating treatmentregimen has had the desired effect. This assay also may be useful in thediagnosis or progression of specific diseases.

In accordance with the invention, an exemplary assay comprises a methodfor assaying the TFR cell function or the TFH cell function or both, ina patient comprising the steps of:

a) Obtaining a sample of peripheral blood from a patient;

b) Isolating a population of TFH cells and TFR cells from the bloodsample;

c) Contacting the TFH cells and TFR cells with a stimulatory compositioncomprising antigen present cells (e.g. B cells) in the presence ofT-cell receptor stimulating factors and cofactors such as anti-CD3 andanti-IgM, for a time period sufficient to allow the production ofantibody such as IgG; and

d) measuring the total antibody produced using standard assays (e.g.ELISA).

VII. Modulation of TFR and TFH Cell-Mediated Immune Responses ViaSelective TFR and TFH Cell Surface Receptors

The data in FIG. 23 shows that TFR cells are distinct in their geneexpression as compared to Treg cells and TFH cells suggesting that TFRcells are capable of independently regulating immune responses. Thisknowledge may be applied to diagnose, monitor and treat diseases orconditions wherein TFR-immune responses may be selectively modulatedsuch as in those diseases or conditions in which antibodies play a keyrole in the pathogenesis and enhanced immune suppression is therapeutic.Examples of such diseases are provided herein supra.

TFR cell function may be modulated by use of an agent such as an agonistor an antagonist of one or more of TFR cell surface receptors asdescribed herein. Use of such an agent in an amount effective to inhibitor induce the differentiation of TFR cells and/or modulate thebiological function of TFR cells can affect the TFR cell-mediated immuneresponse.

In one embodiment, the invention provides a method suppressing apathogenic antibody response in a patient in need thereof comprising,administering to the patient, an agent which modulates at least onereceptor which is differentially expressed on TFR cells as compared toTFH cells at an increased mean fluorescence intensity (MFI) fold changeof at least 1.17, and wherein the receptor has an MFI of at least 186 onTFR cells and wherein the agent administered in an amount that iseffective to modulate the TFR receptor and increase TFR cell-mediatedantibody suppression, as compared to the TFR cell-mediated antibodysuppression in the absence of the agent. Such differentially expressedreceptors are referred to herein as “selective TFR receptors”. In oneembodiment at least one selective TFR receptor is selected from one ormore of: CD162, CD27, CD95, CD9, CD43, CD278 (ICOS), CD50, CD45RB,CD102, CD61, CD58, CD196, CD38, CD31, CD15, CD25, CD13, CD66a/c/e, CD11bCD63, CD32, CD97, HLA-HQ, CD150, Siglec-9, Integrinβ7, CD71, CD180,CD218a, CD193, CD235ab, CD35, CD140a, CD158b, CD33, CD210, HLA-G,CD167a, CD119, CX3CR1, CD146, HLA-DR, CD85, CD172b, SSEA-1, CD49c,CD170, CD66b, and CD86. In one embodiment, selective TFR receptors areselected from one or more of: CD27, CD278 (ICOS), CD150, Siglec-9,CD140a, CD158b, and CD33.

Methods of modulating TFR cell-mediated immune response throughantagonizing or agonizing the biological function of a selective TFRreceptor are useful in the treatment of diseases and conditions whereina decreased or increased TFR cell-mediated immune response is useful.Examples of disorders or conditions which may be treated by increasingTFR cell-mediated immune response include those diseases and conditionsin which antibodies contribute to and/or are primarily responsible for,pathogenesis such as in those diseases listed previously herein. In oneembodiment the disease or condition in which antibodies contributeand/or are primarily responsible for pathogenesis include: multiplesclerosis, systemic lupus erythematosis, allergy, myasthenia gravis,collagen diseases, glomerulonephritis, Devic's disease, vasculitiscaused by ANCA, and celiac disease.

In one embodiment, the agent is a blocking antibody capable ofblocking/antagonizing a selective TFR receptor, or binding to the ligandof the receptor and thereby blocking its ability to bind itscorresponding receptor. The agent may also be a small molecule, or a DNAor RNA molecule (e.g. dsRNA, or antisense molecule) capable ofantagonizing or agonizing the biological function of the receptor, forexample by causing overexpression of a receptor or by blockingexpression of a receptor.

In addition to the known agonists and antagonists of various selectiveTFR receptors known generally, other agents may be tested for theirability to antagonize or agonize selective TFR receptors using knownassays and screens.

Assays and screens of the invention useful for testing various agentsfor their ability to agonize or antagonize TFR cell surface receptorsare used to identify agents of the invention. In one embodiment, theinvention provides assays to specifically and sensitively determine thestimulatory function of TFH cells. Complementary assays are alsoprovided which determine the inhibitory capacity of TFR cells. Theseassays which include both in vitro and in vivo experiments can be usedto determine the functional consequences of sending agonist and/orantagonist signals through surface receptors on TFH and TFR cells.

In vitro murine TFH stimulation assays are performed byimmunizing/vaccinating mice with an antigen/adjuvant. In some cases liveor attenuated virus can be used. Seven to ten days later TFH cellsdefined as (CD4+ICOS+CXCR5+FoxP3+CD19−) or (CD4+ICOS+CXCR5+GITR+CD19−)(or alternative TFH marker) are sorted by flow cytometry. TFH cells areincubated with B cells along with anti-CD3 and anti-IgM (oralternatively with specific antigen). Agonists and/or antagonists forTFH surface receptors are also added into cultures. After 7 daysantibody production and class switch recombination is assessed by eitherstaining B cells from the culture for activation markers (B7-1, GL7,etc.) or intracellular for IgG isotypes. Activation status of the TFHcell can also be determined. Alternatively, the supernatants can beassessed for presence of IgGs via ELISA. Examples of this assay areincluded in FIGS. 4A-4M, 20A-20I and 21A-21E. Similar assays areperformed using human cells isolated from blood or other tissues.

In vivo murine TFH stimulation assays are performed byimmunizing/vaccinating mice with an antigen/adjuvant. In some cases liveor attenuated virus can be used. Seven to ten days later TFH cellsdefined as (CD4+ICOS+CXCR5+FoxP3−CD19−) or (CD4+ICOS+CXCR5+GITR−CD19−)(or alternative TFH marker) are sorted by flow cytometry. Cells areeither used right away, or incubated in vitro with agonists orantagonists as in in vitro assays. Cells are adoptively transferredintravenously to mice that are vaccinated or likewise challenged withantigen and/or virus. Ten days later serum is collected from mice andIgGs are detected by ELISA. Within these 10 days agonists or antagonistsfor TFH surface receptors can be administered.

Examples of this assay are in FIGS. 8A-8K and 21A-21E. These assays canalso be used to determine how TFH cells change disease autoimmunepathology. As an example, mice can be immunized with collagen and thenTFH cells can be sorted and transferred to a new mouse that is immunizedwith collagen. The resulting anti-collagen antibodies will causearthritis which can be measured to determine how TFH cells functionwithin this specific disease. Additionally, these assays can be used todetermine TFH stimulation of B cell antibody production in the contextof vaccination by using TFH cells from influenza infected mice and thenadoptively transfer them to a new mouse that is infected with influenza.Extent of viral infection can be measured as a readout for antibodymediated clearance of virus.

In vitro murine TFR suppression assays are performed byimmunizing/vaccinating mice with an antigen/adjuvant. In some cases liveor attenuated virus can be used. Seven to ten days later TFH cellsdefined as (CD4+ICOS+CXCR5+FoxP3−CD19−) or (CD4+ICOS+CXCR5+GITR−CD19−)(or alternative TFH marker) and TFR cells defined as(CD4+ICOS+CXCR5+FoxP3+CD19−) or (CD4+ICOS+CXCR5+GITR+CD19−) (oralternative TFR marker) are sorted by flow cytometry. TFH and/or TFRcells are incubated with B cells along with anti-CD3 and anti-IgM (oralternatively with specific antigen). Agonists and/or antagonists forTFR surface receptors are also added into cultures. After 7 daysantibody production and class switch recombination is assessed by eitherstaining B cells from the culture for activation markers (B7-1, GL7,etc.) or intracellular for IgG isotypes. Activation status of the TFHcell can also be determined. Alternatively, the supernatants can beassessed for presence of IgGs via ELISA. Examples of this assay areincluded in FIGS. 4A-4M, 20A-20I and 21A-21E. Similar assays areperformed using human cells isolated from blood or other tissues.

In vivo murine TFR suppression assays are performed byimmunizing/vaccinating mice with an antigen/adjuvant. In some cases liveor attenuated virus can be used. Seven to ten days later TFH cellsdefined as (CD4+ICOS+CXCR5+FoxP3+CD19−) or (CD4+ICOS+CXCR5+GITR+CD19−)(or alternative TFH marker) and TFR cells defined as(CD4+ICOS+CXCR5+FoxP3+CD19−) or (CD4+ICOS+CXCR5+GITR+CD19−) (oralternative TFR marker) are sorted by flow cytometry. Cells are eitherused right away, or incubated in vitro with agonists or antagonists asin in vitro assays. Cells are adoptively transferred intravenously tomice that are vaccinated or likewise challenged with antigen and/orvirus. Ten days later serum is collected from mice and IgGs are detectedby ELISA. Within these ten days agonists or antagonists for TFR surfacereceptors can be administered. Examples of this assay are in FIGS. 8A-8Kand 21A-21E. These assays can also be used to determine how TFR cellschange disease pathology. As an example, mice can be immunized withcollagen and then TFH and TFR cells can be sorted and transferred to anew mouse that is immunized with collagen. The resulting anti-collagenantibodies will cause arthritis which can be measured to determine howTFR cells function to suppress the TFH mediated disease.

The present invention further provides agents identified in the assaysdescribed herein. Such agents are capable of up antagonizing oragonizing a selected TFR receptor and thereby modulate TFR cell-mediatedimmune function and to further treat diseases as described herein.

Animal model systems which can be used to screen the effectiveness ofthe selected agents and test agents of the present invention inprotecting against or treating the disease are available. Methods forthe testing of systemic lupus erythematosus (SLE) in susceptible miceare known in the art (Knight et al. (1978) J. Exp. Med., 147: 1653;Reinersten et al. (1978) New Eng. J. Med., 299: 515). Myasthenia Gravis(MG) is tested in SJL/J female mice by inducing the disease with solubleAchR protein from another species (Lindstrom et al. (1988) Adv.Immunol., 42: 233). Arthritis is induced in a susceptible strain of miceby injection of Type II collagen (Stuart et al. (1984) Ann. Rev.Immunol., 42: 233). A model by which adjuvant arthritis is induced insusceptible rats by injection of mycobacterial heat shock protein hasbeen described (Van Eden et al. (1988) Nature, 331: 171). Thyroiditis isinduced in mice by administration of thyroglobulin as described (Maronet al. (1980) J. Exp. Med., 152: 1115). Insulin dependent diabetesmellitus (IDDM) occurs naturally or can be induced in certain strains ofmice such as those described by Kanasawa et al. (1984) Diabetologia, 27:113. EAE in mouse and rat serves as a model for MS in human. In thismodel, the demyelinating disease is induced by administration of myelinbasic protein (see Paterson (1986) Textbook of Immunopathology, Mischeret al., eds., Grune and Stratton, New York, pp. 179-213; McFarlin et al.(1973) Science, 179: 478: and Satoh et al. (1987) J. hnmunol., 138:179).

Generally, suitable agents identified and tested as described above willbe used in purified form together with pharmacologically appropriatecarriers. Typically, these carriers include aqueous or alcoholic/aqueoussolutions, emulsions or suspensions, any including saline and/orbuffered media. Parenteral vehicles include sodium chloride solution,Ringer's dextrose, dextrose and sodium chloride and lactated Ringer's.Suitable physiologically-acceptable adjuvants, if necessary to keep, forexample, a polypeptide complex such as an antibody in suspension, may bechosen from thickeners such as carboxymethylcellulose,polyvinylpyrrolidone, gelatin and alginates. Intravenous vehiclesinclude fluid and nutrient replenishers and electrolyte replenishers,such as those based on Ringer's dextrose. Preservatives and otheradditives, such as antimicrobials, antioxidants, chelating agents andinert gases, may also be present (Mack (1982) Remington's PharmaceuticalSciences, 16th Edition).

The selected agents of the present invention may be used as separatelyadministered compositions or in conjunction with other agents. These caninclude various immunotherapeutic drugs, such as cylcosporine,methotrexate, adriamycin or cisplatinum, and immunotoxins.Pharmaceutical compositions can include “cocktails” of various cytotoxicor other agents in conjunction with the agents of the present invention.

The route of administration of pharmaceutical compositions according tothe invention may be any of those commonly known to those of ordinaryskill in the art. For therapy, including without limitationimmunotherapy, the selected agents can be administered to any patient inaccordance with standard techniques. The administration can be by anyappropriate mode, including parenterally, intravenously,intramuscularly, intraperitoneally, transdermally, via the pulmonaryroute, or also, appropriately, by direct infusion with a catheter. Thedosage and frequency of administration will depend on the age, sex andcondition of the patient, concurrent administration of other drugs,counter indications and other parameters to be taken into account by theclinician.

In certain therapeutic applications, an adequate amount to accomplishmodulation of a TFR and/or TFH cell-mediated immune response will dependupon the severity of the disease and the general state of the patient'sown immune system. Generally, if the agent is a blocking antibody, forexample, a range from 0.01 mg-100 mg per kilogram of body weight, withdoses of 1-10 mg/kg would be suitable.

A composition containing one or more selected agents according to thepresent invention may be used in prophylactic and therapeutic settingsto aid in the modulation of a TFR and/or TFH cell-mediated response. Inaddition, the agents described herein may be used extracorporeally or invitro to selectively modulate TFR and/or TFH cell-mediated immuneresponses.

The invention also provides agents and methods for modulating TFHcell-mediated immune responses. TFH cells are known in the prior art tobe a subset of T helper cells that are genetically distinct from othertypes of T helper cells suggesting that TFR cells may independentlyregulate immune responses. This knowledge may be applied to diagnose,monitor and treat, for example, diseases or conditions whereinenhancement of a protective antibody response is therapeutic. Suchdiseases include but are not limited to treating viral infections andtreating cancer. Enhancing and preferably selectively enhancing, TFHcell-mediated immune responses are also particularly beneficial as partof a vaccine or vaccination regimen.

TFH cell function may be modulated by use of an agent such as an agonistor an antagonist of one or more of TFH cell surface receptors asdescribed herein. Use of such an agent in an amount effective to inhibitor induce the differentiation of TFH cells and/or modulate thebiological function of TFH cells can affect the TFH cell-mediated immuneresponse.

In one embodiment, the invention provides method of increasing aprotective antibody response in a patient in need thereof comprisingadministering to the patient, an agent which modulates at least onereceptor which is differentially expressed on TFH cells as compared toTFR cells at an increased mean fluorescence intensity (MFI) fold changeof at least 1.17, and wherein the receptor has an MFI of at least 200 onTFH cells and wherein the agent is effective to modulate the receptorand increase the antibody response in the patient as compared to theantibody response when the agent is absent. Such differentiallyexpressed receptors are referred to herein as “selective TFH receptors”.In one embodiment at least one selective TFH receptor is selected fromone or more of: CD163, CD127, CD8a, CD89, CD197, CD161, CD6, CD229,CD96, CD272, CD148, CD107a, CD100, CD82, CD126, CD45RO, PD-1 (CD279),CD5, and CD99. In one embodiment, at least one selective TFH receptor isselected from one or more of: CD163, CD127, CD161, CD6, CD229, CD272,CD100, CD126, PD-1 (CD279).

In one embodiment, the agent is a blocking/antagonizing antibody capableof blocking a selective TFH receptor, or binding to the ligand of thereceptor and thereby blocking its ability to bind its correspondingreceptor. The agent may also be a small molecule, or a DNA or RNAmolecule (e.g. dsRNA, or antisense molecule) capable of antagonizing oragonizing the receptor.

In addition to the known agonists and antagonists the various selectiveTFH receptors described herein, other agents may be tested for theirability to antagonize or agonize selective TFH receptors using knownassays and screens. Assays and screens for testing various agents fortheir ability to agonize or antagonize TFH cell surface receptors aredescribed previously herein. Animal models may be used to testmodulation of selective TFH receptors as described previously herein.

The present invention further provides agents identified in the assaysdescribed herein wherein such agents are capable of antagonizing oragonizing a selective TFH receptor and thereby modulate TFHcell-mediated immune function. Agents for modulating selective TFHreceptors may be formulated and administered to patients as describedabove.

In one embodiment, the invention provides a method of decreasing apathogenic antibody response in a patient in need thereof comprisingadministering to the patient a first agent capable of modulating aselective TFR receptor in an amount effective to increase TFRcell-mediated antibody suppression in the patient, in combination withan second agent capable of modulating a selective TFH receptor in anamount effective to decrease TFH cell-mediated antibody production,wherein the pathogenic antibody response is decreased as compared to thepathogenic antibody response in the absence of the first or secondagents.

In one embodiment, the invention provides a method of increasing aprotective antibody response in a patient in need thereof comprisingadministering to the patient a first agent capable of modulating aselective TFR receptor in an amount effective to decrease TFRcell-mediated antibody suppression in the patient, in combination with asecond agent capable of modulating a selective TFH receptor in an amounteffective to increase TFH cell-mediated antibody production, wherein theprotective antibody response is increased as compared to the protectiveantibody response in the absence of the first or second agents.

VIII. Modulation of TFR and TFH Cell-Mediated Immune Responses Via PD-1Receptors

The inventors' discovery that PD-1:PD-L1 interactions limit TFR celldifferentiation and function has also elucidated another novel approachto modulating both TFR cell-mediated and TFH cell-mediated immuneresponses in a patient.

Therefore, in one embodiment the invention provides a method ofdecreasing a pathogenic antibody response in a patient in need thereofcomprising administering to the patient, an agent which modulates thePD-1 receptor on a TFR cell. In one embodiment, the agent is anantagonist of the PD-1 receptor on a TFR cell. In one embodiment, theagent is an antibody capable of blocking the PD-1 receptor on a TFRcell. In one embodiment, the agent is an antibody capable of binding toa ligand selected from PD-L1 or PD-L2 and preventing the ligand frombinding to the PD-1 receptor. The agent may also be a small molecule, ora DNA or RNA molecule (e.g., dsRNA, or antisense molecule) capable ofantagonizing or agonizing the receptor.

In one embodiment a disease or condition wherein suppression of apathogenic antibody response is therapeutic includes those diseaseslisted previously in which antibodies contribute to, or are primarilyresponsible for pathogenesis.

In another embodiment, the invention provides a method of increasing aprotective antibody response in a patient in need thereof comprisingadministering to the patient, an agent which modulates the PD-1 receptoron a TFH cell. In one embodiment the agent is an agonist of the PD-1receptor on a TFH cell. The agent may also be a small molecule, or a DNAor RNA molecule (e.g. dsRNA, or antisense molecule) capable of agonizingthe PD-1 receptor.

In one embodiment, the invention provides a method of decreasing apathogenic antibody response in a patient in need thereof comprisingadministering to the patient a first agent capable of modulating a PD-1receptor on a TFR cell in an amount effective to increase TFRcell-mediated antibody suppression in the patient, in combination withan second agent capable of modulating a PD-1 receptor on a TFH cell inan amount effective to decrease TFH cell-mediated antibody production,wherein the pathogenic antibody response is decreased as compared to thepathogenic antibody response in the absence of the first or secondagents.

In one embodiment, the invention provides a method of increasing aprotective antibody response in a patient in need thereof comprisingadministering to the patient a first agent capable of modulating a PD-1receptor on a TFR cell in an amount effective to decrease TFRcell-mediated antibody suppression in the patient, in combination with asecond agent capable of modulating a PD-1 receptor on a TFH receptor inan amount effective to increase TFH cell-mediated antibody production,wherein the protective antibody response is increased as compared to theprotective antibody response in the absence of the first or secondagents.

In addition to the known agonists and antagonists of the PD1 receptorgenerally, other agents may be tested for their ability to antagonize oragonize PD-1 on TFH and TFR cells using known assays and screens. Assaysand screens for testing various agents for their ability to agonize orantagonize PD-1 receptors are previously described. Animal models may beused to test modulation of selective TFH receptors as described above.

The present invention further provides agents identified in the assaysdescribed herein wherein such agents are capable of antagonizing oragonizing PD-1 receptors and thereby modulate TFR or TFH cell-mediatedimmune function or both.

Agents for modulating PD-1 receptors on TFR or TFH cells may beformulated and administered to patients as described above.

IX. Examples Example 1 Introduction

Follicular helper T cells (TFH) are a recently defined subset of CD4 Tcells that are essential for helping cognate B cells form and maintainthe germinal center (GC) reaction, and for development of humoral immuneresponses. These cells are universally defined by expression of thechemokine receptor CXCR5, which directs them to the B cell follicles viagradients of the chemokine CXCL13¹. TFH cells also express thetranscription factor Bcl6 (which represses Blimp-1/Prdm1) and highlevels of the costimulatory receptor ICOS, which are both critical fortheir differentiation and maintenance¹⁻⁴. In addition, TFH cells secretelarge amounts of IL-21, which aids in GC formation, isotype switchingand plasma cell formation⁵. In humans and mice functionally similar TFHcells can be found in secondary lymphoid organs. Significantly, CXCR5⁺TFH cells are also present in peripheral blood and seen at elevatedlevels in individuals with autoantibodies, including systemic lupuserythematosis, myasthenia gravis and juvenile dermatomyositis patients.However, the function of these circulating TFH remains unclear⁶⁻⁹.

TFH cells also express high levels of programmed death (PD) 1 receptor(CD279). Signaling through PD-1 attenuates TCR signals and inhibits Tcell expansion, cytokine production and cytolytic function. In addition,PD-1 promotes the development of induced regulatory T (iTreg) cells fromnaïve lymphocytes¹⁰⁻¹⁴. PD-1 has two ligands, PD-L1 (B7-H1) and PD-L2(B7-DC). PD-L1 is more widely expressed than PD-L2, but PD-L1 and PD-L2both can be expressed on GC B cells and dendritic cells¹⁵. Perturbationstudies suggest critical roles for this pathway in regulating humoralimmune responses. However, there are conflicting reports as to thefunction of the PD-1 pathway in controlling humoral immunity. Somestudies have found that humoral responses are attenuated¹⁶⁻¹⁸, whileothers have seen that humoral responses are heightened^(19, 20) whenPD-1:PD-L interactions are prevented.

PD-1 also is found on a newly defined subset of CD4⁺CXCR5⁺ cells calledT follicular regulatory (TFR) cells, which are positive for thetranscription factors FoxP3, Bcl6 and Prdm1/Blimp1 and function toinhibit the germinal center response²¹⁻²³. These cells originate fromnatural regulatory T cell precursors, but express similar levels ofICOS, CXCR5 and PD-1 as TFH cells. Since ICOS, CXCR5 and PD-1 have beenwidely used to identify and purify ‘TFH cells’, it seems likely that theinability to define clear functions for PD-1 in GC responses derivesfrom experimental systems containing mixtures of stimulatory TFH cellsand inhibitory TFR cells. We provide a separate analyses of the functionof PD-1 on TFH and TFR cells to elucidate how PD-1 controls humoralimmunity and to gain insight into the individual roles of TFR cells andTFH cells in regulating antibody production.

Here we demonstrate that PD-1:PD-L1 interactions inhibit TFR, but notTFH, cell numbers in lymph nodes. PD-1 deficient mice have increasednumbers of lymph node TFR cells compared to wild type mice. PD-1deficient lymph node TFR cells have enhanced ability to suppressactivation of naïve T cells, as well as antibody production in vitro. Inaddition, we show for the first time that TFR cells are present in theperipheral blood of mice, and that these circulating cells can potentlyregulate humoral immune responses in vivo. Using transfer approaches, wedemonstrate that blood TFH cells can promote antibody production, whileblood TFR cells can strongly inhibit antibody production in vivo. Wefurther show that the PD-1 pathway inhibits blood TFR cell function andPD-1 deficient blood TFR cells have enhanced suppressive capacity invivo. Taken together, our studies reveal a new immunoregulatory role forthe PD-1:PD-L1 pathway in limiting TFR cell differentiation andfunction, and further demonstrate the dynamic control of humoral immuneresponses by migration of TFR cells from the circulation into lymphnodes to control antibody production in vivo.

Methods

Mice.

6-10 week old mice were used for all experiments. WT C57BL/6 andTCRα^(−/−) mice were purchased from The Jackson Laboratory (Bar Harbor,Me.). PD-1^(−/−), PD-L1^(−/−), and PD-L2^(−/−) mice on the C57BL/6background were generated in our laboratory³²⁻³⁴. ICOS^(−/− 35) andCD28^(−/−) mice³⁶ were generated as described. 2D2 TCR Tg miceFoxp3-IRES-GFP knockin mice (Foxp3.GFP;³⁷ were generated in ourlaboratory by crossing 2D2 TCR Tg mice³⁸ with Foxp3.GFP reporter mice.All mice were used according to the Harvard Medical School StandingCommittee on Animals and National Institutes of Animal HealthcareGuidelines. Animal protocols were approved by the Harvard Medical SchoolStanding Committee on Animals.

Immunizations.

For MOG 35-55 immunizations (referred to as “MOG/CFA”), mice wereinjected subcutaneously with 100 μg of MOG 35-55 (UCLA BiopolymersFacility) emulsified in a 1:1 emulsion of H37RA CFA (Sigma) on the mouseflanks. Seven days later mice were euthanized and inguinal lymph nodes(dLN) and/or spleen were harvested for flow cytometric analyses. Bloodwas collected via cardiac puncture with a 1 cc syringe and immune cellswere isolated by sucrose density centrifugation using LymphocyteSeparation Media (LSM). For NP-OVA immunizations, 100 μg NP₁₈-OVA(Biosearch Technologies) was used in a 1:1 H37RA CFA emulsion andinjected similarly as MOG/CFA.

ELISA.

For in vitro quantitation of antibody production, supernatants weretaken from cultures and total IgG was quantified using pan-IgG captureantibody (Southern Biotech) and alkaline phosphatase conjugated pan-IgGdetection antibody (Southern Biotech). To assess in vivo antibodyproduction, sera were collected from mice at indicated time points.NP-specific antibody titers were measured by coating ELISA plates withNP₁₆-BSA (Biosearch Technologies), and incubating serum for 1 hrfollowed by alkaline phosphatase-conjugated IgG detection antibodies. Astandard curve was generated using antibody from an NP-specific IgG1hybridoma (a kind gift of Dr. Michael Carroll). This standard curve wasused to approximate all IgG subtype antibody levels in the linear rangeof detection using a Spectramax Elisa plate reader (Molecular Devices).

Flow Cytometry.

Cells from lymphoid organs were isolated and resuspended in stainingbuffer (PBS containing 1% fetal calf serum and 2 mM EDTA) and stainedwith directly labeled antibodies from Biolegend against CD4 (RM4-5),ICOS (15F9), CD19 (6D5), PD-1 (RMP1-30), PD-L1 (10F.9G2), CD69 (H1.2F3),from eBioscience against FoxP3 (FJK-165), Bcl6 (mGI191E), and from BDbioscience against FAS (Jo2), GL7, Ki67 (B56). For CXCR5 staining,biotinylated anti-CXCR5 (2G8, BD Biosciences) was used followed bystreptavidin-brilliant violet 421 (Biolegend). For intracellularstaining, the FoxP3 fix/perm kit was used (eBioscience) after surfacestaining was accomplished. All flow cytometry was analyzed with an LSRII (BD biosciences) using standard filter sets.

Confocal Microscopy.

Draining lymph nodes were embedded in OCT and 8 μm sections were cut,fixed with 4% paraformaldehyde, and stained before imaging on a ZeissLSM 510 confocal microscope by acquiring z-stacks of 0.5 um with a 40×oil objective. Germinal center quantitation was calculated by drawingoutlines around GL7⁺ IgD⁻ areas present within the B cell zone. FoxP3quantitation was performed by determining germinal center zones andscrolling through z-stack images to identify large FoxP3 positive spots.Axiovision (Zeiss) software was used to measure distances from germinalcenter borders. For micrograph panels, single z slices were linearlycontrasted and merged images were made in Adobe photoshop.

Quantitative PCR.

Q-PCR was performed using standard TaqMAN probes (Applied Biosystems)and an ABI FAST9500 QPCR machine according to the manufacturer'sinstructions. mRNA levels were normalized to HPRT or β2M, and the2^(−deltadeltaCT) method was used to quantitate mRNA. Each bar graphrepresents mean values from more than three individual experimentsconsisting of cells sorted from 5-10 pooled mice.

In Vitro Suppression Assay.

Cell populations were sorted to 99% purity on an Aria II flow cytometer.For TFR suppression assays, sorted cells were counted on an Accuricytometer (BD biosciences) by gating live cells only, and 1×10⁵ GL7⁻ Bcells from dLNs of WT mice immunized with MOG/CFA 7 days previously,1×10⁵ CFSE labeled CD4⁺CD62L⁺FoxP3⁻ T responder cells from unimmunizedWT FoxP3 GFP reporter mice, and 1×10⁵ TFR cells from the draining dLN of10 pooled mice immunized with MOG/CFA 7 days previously were plated with2 μg/ml soluble anti-CD3 (2C11, BioXcell) and 5 μg/ml anti-IgM (JacksonImmunoresearch). After 3 days, cells were harvested and stained for CD4and CD19. T cell responders were identified as CFSE positive, andpercent divided was gated as the percent of cells with CFSE dilutedcompared to unstimulated T responders.

Adoptive Transfers.

For blood TFH/TFR adoptive transfers, 20 to 30 WT mice were immunizedwith NP-OVA subcutaneously as described above, and 8 days later bloodwas collected by cardiac puncture. TFH and TFR cells were sorted asdescribed. 4×10⁴ blood TFH cells alone or together with 2×10⁴ blood TFRcells were transferred into CD28^(−/−) or TCRα^(−/−) mice unlessspecified otherwise. These recipient mice were immunized with NP-OVA asdescribed above. Serum and organs were harvested at indicated times andanalyzed by ELISA or flow cytometry.

Statistical Analysis.

Unpaired Student's t test was used for all comparisons, data representedas mean+/−SD or SE are shown. P values <0.05 were consideredstatistically significant. * P<0.05, ** P<0.005, *** P<0.0005.

Results PD-1 Controls T Follicular Regulatory Cells

To analyze the role of PD-1 in controlling T follicular regulatory (TFR)cells, we first compared PD-1 expression on CD4 T cell subsets indraining lymph nodes (dLN) of WT C57BL/6 mice subcutaneously immunizedwith myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 emulsifiedin CFA (from herein simply referred to as “MOG/CFA”), an immunizationthat breaks tolerance and also results in effective TFH cellgeneration²⁴. TFR cells were defined as CD4⁺ICOS⁺CXCR5⁺FoxP3⁺CD19⁻, agating strategy that separates TFR cells from CD4⁺ICOS⁺CXCR5⁺FoxP3⁻CD19⁻TFH cells, the cell type that was until recently thought to solelycomprise the CD4⁺CXCR5⁺gate (FIG. 1A). TFH cells showed higherexpression of PD-1 compared to ICOS⁺CXCR5⁻ effector-like cells andICOS⁻CXCR5⁻ naïve (referred to as naïve) cells in the draining lymphnode on day 7 after immunization. Strikingly, TFR cells had even higherPD-1 expression when compared to the other CD4 T cell subsets examined,including TFH cells (FIG. 1B).

To determine the functional significance of PD-1 expression on TFRcells, we immunized WT and PD-1^(−/−) mice and analyzed TFR cells 7 dayslater. The percentage of TFR cells contained within the CD4⁺FoxP3⁺ gatewas about 4 percent in WT lymph nodes and less than 1 percent of all CD4T cells. In marked contrast, the percentage of TFR cells in PD-1^(−/−)mice was about 10 percent of the CD4⁺FoxP3⁺ gate and greater than 2percent of all CD4 T cells (FIGS. 1C-D). Because total numbers of CD4 Tcells are typically about two fold higher in the lymph nodes of PD-1deficient animals, a two-fold increase in TFR cell frequency translatesinto a ˜4-fold increase in absolute numbers of TFR cells (data notshown). When expressed as a percentage of all CD4⁺ICOS⁺CXCR5⁺ cells (andtherefore the percentage of CD4 T cells that respond to CXCL13 andmigrate to the B cell zone), PD-1^(−/−) TFR cells comprised half of thispopulation, whereas WT TFR cells comprised only about 20 percent (FIG.1D). The dramatic increase in the percentage of TFR cells in PD-1^(−/−)mice also was observed when other classical B cell antigens, such as4-hydroxy-3-nitrophenylacetyl hapten conjugated to ovalbumin (NP-OVA),were used (FIG. 10A-10E). We did not find a significant difference inthe percentage of FoxP3⁻ TFH (from hereon called “TFH”) cells whenexpressed as a percentage of all CD4 T cells in WT and PD-1^(−/−) miceon day 7 post immunization (FIG. 1E).

Since PD-1 can be expressed by a number of hematopoietic cell typesincluding T cells, B cells, macrophages and some dendritic cells¹⁵, wenext investigated whether PD-1 regulates TFR cells directly bycontrolling their generation from FoxP3⁺ T regulatory cells (Treg). Totrack the fate of FoxP3⁺ cells following transfer into WT mouserecipients, we used antigen-specific FoxP3⁺ T cells from TCR transgenicmice for these studies. We sorted FoxP3⁺ Tregs from WT or PD-1^(−/−) 2D2(MOGspecific) TCR transgenic FoxP3 GFP reporter mice and transferred2×10⁵ 2D2 WT or PD-1^(−/−) CD4⁺CXCR5⁻FoxP3⁺ cells into WT recipientmice. We immunized these recipients with MOG/CFA and analyzed cells inthe draining lymph node seven days later (FIG. 1F). There were a greaterpercentage (FIG. 1H) and absolute number (FIG. 1I) of 2D2 PD-1^(−/−)Tregs upregulating CXCR5 and thus differentiating into TFR cells,compared to 2D2 WT Tregs. The increased percentage of PD-1^(−/−) TFRcells in the immunized transfer recipients was similar, but lesspronounced, than the increased percentage in TFR cells seen in immunizedintact PD-1 deficient mice (FIG. 1D, H). These results demonstrate thatPD-1 controls differentiation of FoxP3⁺ Tregs into TFR cells.

Since CD25 (the alpha chain of the IL-2 receptor) is frequently used asa marker for Tregs, we next compared CD25 expression on WT andPD-1^(−/−) TFR cells directly ex vivo (FIG. 2A). PD-1^(−/−) TFR cellsexpressed less CD25 than WT TFR cells (FIG. 2B). The attenuated CD25expression in PD-1 deficient TFR cells is not likely due to decreasedactivation because the expression of the early activation marker CD69was virtually identical on WT and PD-1^(−/−) TFR cells (FIG. 2C). Tocompare the proportion of WT and PD-1^(−/−) TFR cells proliferating atday 7 post immunization, we examined Ki67 expression, a marker widelyused to identify cells that are actively dividing. WT ICOS⁺ CXCR5⁻effectors, TFH and TFR gated cells had high expression of Ki67. Incontrast, the WT CXCR5⁻ICOS⁻ “naïve” cells, lacking CD69 and CD25expression, had no Ki67 staining consistent with their designation asnaïve (FIG. 2D). WT TFR cells expressed significantly higher levels ofKi67 compared to PD-1^(−/−) TFR cells, suggesting that the increasednumbers of TFR cells in PD-1 deficient animals reflect increaseddifferentiation, and not maintenance, of TFR cells. Ki67 expression wassimilarly greater in WT ICOS⁺ effectors and TFH cells compared toPD-1^(−/−) ICOS⁺CXCR5⁻ effectors and TFH cells. These points to anoverall decrease in cell cycling in PD-1^(−/−) effector cells at 7 daysafter immunization. Other Treg markers such as CD103 and GITR were notaltered on TFR cells in PD-1 deficient mice (FIG. 11A-11C).Additionally, there was low, but significant expression of PD-L1 on WTand PD-1^(−/−) TFR cells. Together, these data indicate that PD-1 isimportant in regulating numbers of TFR cells in vivo.

PD-1 Deficient TFR Cells are Capable of Homing to Germinal Centers

We next compared the capacity of TFR cells from WT and PD-1 deficientanimals to enter the germinal center (GC) in order to inhibit the GCresponse. First, we evaluated GC formation in lymph node sectionsharvested 7 days after MOG/CFA immunization. GCs were identified by thepresence of PNA/GL7 positively stained and IgD negatively stained B cellzones (FIG. 3A). These GCs were determined to be active, based on robustexpression of the cell cycle marker Ki67 (FIG. 3B). Similar to previousreports^(21, 22), CD4⁺FoxP3⁺ TFR cells could be found within GCs ofimmunized mice (FIG. 3C). The FoxP3 protein within the TFR cells wasjudged to be largely nuclear based on its co-localization with the DAPIstaining (FIG. 3D).

We then investigated whether the phenotypically distinct TFR cells fromPD-1 deficient mice were able to migrate to GCs similarly to WT TFRcells, because PD-1 blockade can prolong the TCR stop signal anddecrease T cell migration²⁵. We immunized WT and PD-1 deficient micewith MOG/CFA and 7 days later analyzed lymph node sections for IgD, GL7and FoxP3 expression (FIG. 3E). Although the average germinal centerarea (FIG. 3F) and numbers of germinal centers per lymph node (data notshown) were equivalent in WT and PD-1^(−/−) mice, there were slightlymore FoxP3⁺ cells (and therefore TFR cells) located within the GCborders in PD-1^(−/−) mice as in WT mice (FIG. 3G). However, since thisincrease is proportional to the larger numbers of TFR cells in PD-1deficient mice determined by flow cytometry, these data demonstrate thatPD-1 deficient TFR cells are not defective in homing to GCs and canenter the GC similarly to WT TFR cells.

The relative location of FoxP3⁺ TFR cells within the GC did not differsignificantly between WT and PD-1^(−/−) TFR cells (FIG. 3H). In both WTand PD-1^(−/−) mice the FoxP3⁺ cells tended to reside close to the GCborder, with more than half of the FoxP3⁺ nuclei being positioned within10 μm of the border. Furthermore, when CXCR5 fluorescence was quantifiedby flow cytometry in TFR cells, there was similar CXCR5 expression onTFR cells in the WT and PD-1^(−/−) mice, indicating similar potentialfor these cells to respond to chemokine cues to migrate to GCs (FIG.3I). Taken together, these data indicate that TFR cells are increased inlymph nodes of immunized PD-1^(−/−) mice, and these PD-1^(−/−) TFR cellsare capable of migrating into GCs to regulate B cell responses.

PD-1 Deficient TFR Cells More Potently Inhibit T Cell Activation

We next compared the function of TFR cells from WT and PD-1^(−/−) mice.TFR cells express higher levels of GITR on the cell surface than do TFHcells, which allows for separation of the TFH and TFR cells in a similarmanner to intracellular staining for FoxP3 (FIG. 4A). For functionalstudies, we sorted TFR cells from immunized mice by taking the lymphnode CD4⁺ICOS⁺CXCR5⁺CD19⁻GITR⁺ population as TFR cells and theCD4⁺ICOS⁺CXCR5⁺CD19⁻GITR⁻ population as TFH cells (FIG. 4B). Sorting inthis fashion shows robust mRNA for FoxP3 in the GITR⁺ (TFR) population,but essentially no FoxP3 mRNA in the GITR⁻ (TFH) population, validatingthe use of this gating strategy to isolate TFR and TFH cells forfunctional assays. Furthermore, this sorting strategy can be used tocompare WT and PD-1 deficient TFR cells since GITR expression isidentical on WT and PD-1 deficient TFR cells (FIG. 12).

TFR cells express high Blimp1/Prdm1 and moderate levels of Bcl6²¹. Bcl6and Blimp1 reciprocally modulate each other²; Bcl6 inhibition of Blimp1is essential for maintenance of the TFH phenotype, whereas Blimp1 isimportant in Treg homeostasis in general^(26, 27). Since relativeexpression of Bcl6 and Blimp1 determines function of TFH subsets, wecompared Bcl6 expression in TFR cells from WT and PD-1^(−/−) mice usingflow cytometry to analyze intracellular Bcl6 expression at the proteinlevel. Although TFR cells expressed less Bcl6 at the protein level thanTFH cells, WT and PD-1^(−/−) TFR had similar Bcl6 levels (FIG. 4C). Wenext compared the expression of Blimp1 (encoded by Prdm1) on TFR cellsfrom WT and PD-1^(−/−) mice. At the mRNA level, we did not find anyconsistent differences in Blimp1/Prdm1 expression between WT andPD-1^(−/−) TFR cells (FIG. 4D). Since FoxP3 can directly interact withand negatively regulate the function of Rorγt²⁸, we also examined Rorc(which encodes Rorγt) in WT and PD-1^(−/−) TFR cells. Rorc mRNA levelswere lower in TFR cells compared to TFH cells, but Rorc expression wasincreased in PD-1^(−/−) TFR cells relative to WT TFR cells (FIG. 4E). Inaddition, we compared expression of the transcription factor IRF4 in WTand PD-1^(−/−) TFR cells, since Blimp1 and IRF4 synergistically controlthe differentiation and effector functions of regulatory T cells²⁶. Wefound an increase in IRF4 mRNA in PD-1^(−/−) TFR cells compared to WTTFR cells (FIG. 4F).

IRF4 is essential for the suppressive capacity of regulatory T cells²⁶.To determine if increased IRF4 mRNA in PD-1^(−/−) TFR cells translatesinto an increase in suppression of naïve T cell proliferation, we set upan in vitro suppression assay in which we cultured sorted WT GL7⁻ Bcells, CFSE labeled WT naïve CD4⁺CD62L⁺FoxP3⁻ responder T cells, andeither WT or PD-1^(−/−) TFR cells sorted from mice immunized withMOG/CFA together with anti-CD3 and anti-IgM (FIG. 4G). The responder Tcells highly upregulated CD69 after 3 days of culture with WT B cells.However, when WT TFR cells were added in a 1:1:1 ratio, the CD69expression on the responder T cells was much lower, consistent with thefunction of TFR cells in suppressing T cell activation (FIG. 4H). CD69upregulation was inhibited to an even greater extent in responder Tcells that were cultured with PD-1^(−/−) TFR cells. Moreover, PD-1^(−/−)TFR attenuated the proliferation of responder T cells (FIG. 4I), incontrast to WT TFR cells, which did not inhibit the proliferation ofresponder T cells during the day 3 culture period.

Although TFR cells are thought to inhibit the germinal response in vivo,it is unclear whether TFR cells directly inhibit T cell differentiation,TFH cell function, B cell activation or all three. To assess thecapability of TFR cells to suppress B cell antibody production in vitro,we cultured WT GL7⁻ B cells with WT FoxP3⁻ TFH cells for 6 days in thepresence or absence of TFR cells, anti-IgM and anti-CD3 (FIG. 4J). WT Bcells produced large amounts of IgG when cultured with WT FoxP3⁻ TFHcells plus anti-IgM and anti-CD3 (FIG. 4K). No significant IgG waspresent when CD4⁺FoxP3⁻ naïve T cells were used in these experiments(FIG. 4L). When TFR cells were added to the wells along with TFH cells,almost no IgG was produced. The TFR-mediated suppression was not due tosequestering of anti-CD3 because there was equally good suppression atthe two doses of anti-CD3 tested (FIG. 4K), and the anti-CD3 could stillbe found on the surface of the TFH cells at the end of the suppressionassay (FIG. 13A-FIG. 13B). PD-1^(−/−) TFR cells suppressed IgGproduction more than WT TFR cells at both a 1:1 (FIG. 4L) and a 1:5(FIG. 4M) TFR:TFH ratio, with PD-1 deficient TFR cells resulting in a50% greater reduction in IgG production compared to WT TFR cells. Takentogether, these data demonstrate not only that are there increased TFRcells in PD-1^(−/−) mice, but that these PD-1^(−/−) TFR cells haveincreased suppressive capacity.

PD-1 Controls Blood T Follicular Regulatory Cells

One possible explanation for the increase in TFR cells in lymph nodes ofimmunized PD-1 deficient mice is that PD-1^(−/−) TFR cells are unable toexit the lymph node. Studies have demonstrated that functional TFH cellscan be found in the blood of humans as well as mice^(6, 7, 9), butwhether TFR cells circulate in the blood of humans or mice is not yetknown. Strikingly, we found a significant population of TFH cells, aswell as a smaller population of TFR cells, in the blood of WT miceimmunized with MOG/CFA (FIGS. 5A-B). When we compared the kinetics ofTFH and TFR cell expansion in the lymph node and blood of mice followingMOG/CFA immunization, we found that both TFR and TFH cells increase inthe draining lymph node of WT immunized mice over a 10 day period, andthat TFH cells, but not TFR cells, increase substantially by percentagein the blood over this time (FIG. 5B). Thus, without antigenic stimulus,the blood TFR:TFH ratio is fairly high (sometimes greater than 1:1) butupon addition of a stimulus, blood TFH cells expand more than blood TFRcells so that the TFR:TFH ratio is about 1:5. To investigate whether WTblood TFH and TFR cells are quiescent or are actively in cell cycle, wecompared Ki67 expression in draining lymph node and blood TFH and TFRcells 7 days after immunization. TFH cells from the draining lymph nodehad higher Ki67 expression than those found in the blood (FIG. 5C).Blood TFH and TFR and draining lymph node TFR cells expressed similarlevels of Ki67.

Next we investigated whether TFR cells in the blood were inhibited tothe same degree by PD-1 signaling as lymph node TFR cells. We immunizedWT and PD-1^(−/−) mice with MOG/CFA and 7 days later analyzed the bloodfor TFH and TFR cells. In WT mice ˜2-3 percent of CD4⁺FoxP3⁻CD19⁻ cellsin the blood were TFH cells, but in the PD-1^(−/−) mice this increasedto ˜4-5 percent (FIG. 5D). This increase in PD-1^(−/−) TFH cells inblood markedly contrasts with the lymph node, where PD-1^(−/−) mice havesimilar, if not less, TFH cells compared to WT mice (FIG. 1E).Importantly, TFR cells comprised ˜3 percent of all FoxP3 positive cellsin the blood of WT mice, but more than 7 percent of FoxP3 positive cellsin the blood of FD-1^(−/−) mice (FIGS. 5D-E). The increase in FoxP3⁺cells seems to be specific to the blood TFR subset, as the percentage ofFoxP3⁺ cells in the ICOS⁺CXCR5⁻ (ICOS⁺) and ICOS⁻CXCR5⁻ naïve cell gateswere not increased in PD-1^(−/−) mice (FIG. 5F). Taken together, thesedata indicate that both TFR and TFH cells are present in the blood ofmice, and both subsets are repressed by PD-1 signals.

To investigate whether blood TFH and TFR cells have a central memoryphenotype, we analyzed surface expression of CD62L and CD44. About 60%of WT and PD-1^(−/−) blood TFR cells had high expression of CD62L (FIG.14A). This contrasts with the greater than 90% of ICOS⁻CXCR5⁻ naïvecells that had high CD62L expression. PD-1^(−/−) TFH cells had lowerCD62L compared to WT TFH cells. CD44 was highly expressed on all WT andPD-1^(−/−) blood TFR cells, but PD-1^(−/−) blood TFR cells had slightlylower surface expression (FIG. 14B). Furthermore, PD-1 was expressed atlower levels on blood TFR cells than lymph node TFR cells (FIG. 14C).Taken together, these data indicate that blood TFR cells can have acentral memory homing phenotype.

The increase in TFR cells in FD-1^(−/−) mice led us to investigate whichPD-1 ligand is critical for controlling lymph node and blood TFRgeneration. We first compared PD-L1 and PD-L2 expression on B cells andDCs present in dLNs of immunized WT mice because both B cells anddendritic cells (DCs) contribute to proper TFH differentiation andmaintenance in the lymph node¹. It is not yet clear whether B cells, DCor both are needed for TFR differentiation and/or maintenance. To studyGC B cells, we immunized mice with NP-OVA subcutaneously and 12 dayslater compared PD-L1 and PD-L2 expression on FAS⁺GL7⁺CD19⁺ GC B cells,as well as CD138⁺ positive plasma cells (PC) from the dLN. We found thatall B cell subsets expressed high levels of PD-L1, but only GC B cellsexpressed high levels of PD-L2 (FIG. 6A). To quantify PD-L1 and PD-L2expression on DCs, we immunized mice with NP-OVA and analyzed DCpopulations from the draining lymph node 3 days later. Both CD8α⁺ andCD8α⁻DC populations expressed high levels of PD-L1 and moderate levelsof PD-L2 (FIG. 6B). A subpopulation of CD8α⁻ DCs expressed high levelsof PD-L2.

To determine which ligand is important for TFH and TFR generation, weimmunized WT, PD-L1^(−/−) and PD-L2^(−/−) mice with MOG/CFA, andanalyzed TFH and TFR cells 7 days post-immunization in the draininglymph node and blood. The percentages of lymph node TFH cells inPD-L1^(−/−) and PD-L2^(−/−) mice were comparable to WT mice (FIG. 6C)and PD-1^(−/−) mice (FIG. 1E). PD-L1^(−/−), but not PD-L2^(−/−) mice,had greater blood TFH cell numbers, which was similar to PD-1^(−/−) mice(FIG. 5E). TFR cells, however, were increased in the lymph nodes as wellas the blood of PD-L1^(−/−), but not PD-L2^(−/−) mice (FIG. 6D). Similarto PD-1^(−/−) mice, PD-L1^(−/−) mice did not exhibit any increases innon-TFR FoxP3⁺ effector cells within the blood (FIG. 6E). These studiesdemonstrate that PD-L1, but not PD-L2, is responsible for controllinglymph node and blood TFR cells.

Blood TFH and TFR Cells Require CD28 and ICOS Signals

We further investigated the costimulatory requirements for blood TFRcells, focusing on the effects of CD28 and ICOS costimulation onCD4⁺ICOS⁺CXCR5⁺FoxP3⁺CD19⁻ TFR populations in the blood due to theimportant roles of these costimulatory receptors in controlling lymphnode TFH and TFR cells. CD28^(−/−) mice are deficient in lymph node TFHand TFR cells²¹. ICOS^(−/−) mice are deficient in lymph node TFHcells²⁴. We analyzed CD28 and ICOS deficient animals for the presence ofTFR cells in the lymph nodes and blood 7 days after immunization withMOG/CFA. In WT mice, there were fewer TFH and TFR cells in the bloodcompared to the draining lymph node (FIGS. 7A-B). Numbers of TFR (andTFH) cells were greatly attenuated in the blood, as well as lymph nodes,of ICOS deficient mice (FIGS. 7A-C). CD28 deficient mice had similarsevere deficiencies in TFR and TFH cell percentages in both lymph nodesand blood (FIGS. 7E-F). Thus, ICOS and CD28 supply essentialcostimulatory signals for TFR and TFH cells in the blood as well as thelymph nodes.

PD-1 Deficient Blood TFR Cells More Potently Regulate AntibodyProduction In Vivo

We next investigated the function of blood TFR cells in humoral immuneresponses. Because TFH cells in human blood can function in B cellactivation and antibody production in vitro^(6, 7), we analyzed whethercirculating blood TFR cells contribute to suppression of antibodyproduction in vivo. To assess this, we designed transfer experiments inwhich we immunized >20 WT donor mice with NP-OVA subcutaneously and 8days later sorted TFR (CD4⁺ICOS⁺CXCR5⁺GITR⁺CD19⁻) and TFH(CD4⁺ICOS⁺CXCR5⁺GITR⁻CD19⁻) cells from the blood (FIG. 8A). Wetransferred these cells into CD28^(−/−) or TCRα^(−/−) mice because theylack both blood and lymph node TFH and TFR cells. This approach enabledus to determine if blood TFR and TFH cells could regulate humoralresponses. Since the transferred blood TFH and TFR cells are the onlyfollicular T cells in CD28^(−/−) and TCRα^(−/−) recipients, anyresponses in the draining lymph node would be due to trafficking of theblood TFR and TFH cells.

Initially, we adoptively transferred 4×10⁴ TFH cells alone or togetherwith 2×10⁴ TFR cells into CD28^(−/−) mice (approximately a two-foldhigher ratio of TFR:TFH cells than is found in blood afterimmunization). We immunized recipients 1 day later with NP-OVA andanalyzed NP-specific IgG titers 12 days after immunization (FIG. 8A).Without blood TFH or TFR cell transfer, CD28^(−/−) mice were unable toproduce significant amounts of NP-specific IgG (FIG. 8B). The transferof blood TFH cells alone resulted in a substantial increase inNP-specific IgG titers. Transfer of blood TFH cells led to substantialproduction of IgG1, but also smaller increases in other isotypes (datanot shown). Significantly, transfer of blood TFR cells along with bloodTFH cells resulted in robust inhibition of NP-specific antibodyproduction, demonstrating the potent regulatory capacity of blood TFRcells in suppressing antibody production (FIG. 8B). To evaluate theimpact of TFR cells on plasma cell generation, draining lymph nodes,spleens and bone marrow were harvested 24 days after immunization andplasma cells were quantified. CD138⁺ plasma cells were absent from thelymph nodes of immunized CD28^(−/−) mice (FIGS. 8C-D). Transfer of bloodTFH cells resulted in a sizable population of plasma cells in thedraining lymph node, spleen and bone marrow (FIGS. 8C-D). Blood TFRcells almost completely prevented plasma cell formation/survival in allorgans tested.

Next we transferred blood TFH and/or TFR cells into TCRα^(−/−)recipients. Transfer of 4×10⁴ TFH cells resulted in high levels ofNP-specific IgG and at a greater titer than an immunized WT mouse inmost experiments (FIG. 8E). The robust antibody production elicited byblood TFH cells depends on the “follicular program” because transfer oftotal CD4 T cells from CXCR5^(−/−) mice or CD4⁺CXCR5⁻FoxP3⁻ naïve cellsresulted in near background levels of antibody production in theseexperiments (FIG. 8E). When blood TFR cells were transferred togetherwith blood TFH cells, the NP-specific antibody titers were markedlyreduced, demonstrating the suppressive capacity of these cells (FIG.8E). The blood TFR cells resulted in both lower plasma cell percentages(FIG. 8F), as well as lower percentages of TFH cells within the lymphnode (FIG. 8G). When we compared the functions of blood TFH cells anddraining lymph node TFH cells following transfer into TCRα^(−/−)recipients, we found that blood TFH cells have an increased capacity topromote NP-specific IgG production (FIG. 8H). TFR suppression of TFHcells also depends on the “follicular program” in these cells becauseneither blood CD25⁺CD62L⁺ Tregs from CXCR5^(−/−) mice (data not shown)nor blood CXCR5⁻ FoxP3 GFP⁺ Tregs from FoxP3 reporter mice possess thesame suppressive capacity as WT blood TFR cells (FIG. 8I).

Finally, we investigated the suppressive capacity of PD-1 deficientblood TFR cells in vivo since we have found that PD-1 deficient lymphnode TFR cells more potently suppress antibody production in vitro. Weadoptively transferred 4×10⁴ blood TFH cells alone or together with1.5×10⁴ blood TFR cells from WT or PD-1 deficient mice into eitherCD28^(−/−) or TCRα^(−/−) recipients and immunized as in FIG. 8A. PD-1deficient TFR cells inhibited antibody production to a greater extentthan WT TFR cells in CD28^(−/−) (FIG. 8J) as well as TCRα^(−/−) (FIG.8K) recipients, demonstrating that they have increased suppressivecapacity. Together, these data show that blood TFR cells potentlyinhibit antibody production in vivo and PD-1 deficiency results inenhanced TFR cell suppressive capacity.

Discussion

A mechanistic understanding of TFR cell differentiation and function isprovided to gain insight into how humoral immune responses are regulatedby TFH and TFR cells. Although TFR cells originate from differentprecursors than TFH cells, TFR and TFH cells have nearly identicalsurface receptors. The shared expression of ICOS, CXCR5 and PD-1 by TFRand TFH cells means that functional studies of TFH cells have, in fact,examined mixtures of stimulatory TFH cells and inhibitory TFR cells. ThePD-1 pathway regulates many effector arms of the immune response howeverbiological complexity has led to inconsistencies regarding the role ofthis pathway in humoral immune responses¹⁶⁻²⁰. In this Example 1, weidentify a new mechanism by which PD-1 regulates humoral immunity: PD-1controls the generation and function of suppressive TFR cells. We foundthat lack of PD-1, or its ligand PD-L1, resulted in greater numbers ofTFR cells in the draining lymph node of immunized mice. These PD-1^(−/−)lymph node TFR cells expressed more IRF4 and showed an enhanced abilityto suppress antibody production. We also discovered that TFR cells arepresent in the blood of mice, and that PD-1 controls the numbers ofblood TFR cells, as evidenced by the substantial increases in PD-1deficient mice. Importantly, we demonstrated a functional role for bloodTFH cells in promoting antibody production and blood TFR cells insuppressing antibody production in vivo. PD-1 deficient blood TFR cellsmore potently suppress antibody production compared to WT blood TFRcells. Thus, PD-1 limits the development and function of TFR cells inlymph nodes and in the circulation.

We have found that PD-1 signaling inhibits the numbers of TFR cells, butnot TFH cells, in the lymph node, skewing the TFR to TFH ratio. It ispossible that the greater suppressive capacity of PD-1^(−/−) TFR cells,together with the increased ratio of TFR to TFH cells in PD-1^(−/−)mice, results in inhibition of PD-1^(−/−) TFH cells. Alternatively,there may be alterations in PD-1^(−/−) TFH cells that promote theirdeparture from the lymph node and homing to other sites to performeffector functions. These hypotheses are not mutually exclusive. Somestudies have described increases in lymph node/spleen TFH cells in PD-1deficient mice; however the contribution of TFR cells in these studieswas not assessed^(16, 18, 19). It is likely that increased TFR cells inPD-1/PD-L1 knockout mice may have contributed to the increases in TFHcells observed in these studies, and may explain, at least in part,conflicting data regarding the role of PD-1 in regulating TFH cells andgerminal center reactions. For example, Kawamoto et al. describedincreased CD4⁺CXCR5⁺ (TFH) cells in the Peyer's patches of PD-1deficient mice. However, upon transfer, these cells were non-functionalin supporting IgA production. Increases in TFR cells contained withinthe TFH gate in PD-1 deficient Peyer's patches may explain these data.

TFR cells depend on SAP, CD28 and Bcl6 for differentiation^(21, 22).However, the pathways that limit TFR cell differentiation are lessclear. To date, only the transcription factor Blimp1 has been shown toinhibit TFR cell differentiation²¹. Here we identify PD-1 as the firstsurface receptor that inhibits TFR cell development and function. Wealso show that PD-1 predominantly interacts with PD-L1, and not PD-L2,to inhibit TFR generation. Our adoptive transfer studies demonstrate acell intrinsic role for PD-1 in TFR cell differentiation from FoxP3⁺Treg cells. Therefore, the increase in TFR cells in PD-1 deficient miceappears to reflect increased differentiation and not maintenance. Weobserved a general trend for a decrease in cell cycling of CD4PD-1^(−/−) effector cells, because ICOS⁺CXCR5⁺FoxP3⁻ and ICOS⁺CXCR5⁻effector cells also had diminished Ki67 expression, at least at day 7after immunization, which may temporally correspond to a maintenancephase. Based on these data, we believe that, rather than simplyinhibiting responses, the PD-1:PD-L pathway can act as a molecularswitch that controls cell fate decisions in naïve CD4 T cells.Integration of signals through PD-1, the TCR and cytokine receptors maydirect CD4 T cell subset differentiation. Likewise, PD-1 may limitdifferentiated effector T cell expansion, cytokine production and/orsurvival depending on how signals through the TCR, PD-1 and cytokinereceptors are integrated. Thus, the PD-1 pathway can influence CD4 Tcell lineage commitment in distinct ways, depending on molecular cuesand the local environment. For instance, PD-L1 can promote induced Treg(iTreg) differentiation from naïve T cell precursors^(10-14, 29).However, we find that PD-1 inhibits differentiation of TFR cells. TFRcells arise from natural T regulatory cell (nTreg) precursors (shownhere and previously²¹). Therefore, our studies suggest that PD-1 mayhave distinct roles in iTreg and nTreg differentiation. In addition,genetic background may contribute to the effects of PD-1 deficiency.

Because of the recent discovery of TFR cells, there is a lack offundamental knowledge about TFR cell biology, so we developed novelassays to analyze mechanisms by which PD-1 regulates TFR cell function.TFR cells have the potential to directly inhibit activation of naïve Tcells, TFH cell function, and/or B cell activation. TFR cells mightregulate TFH or B cell responses either inside the B cell follicleand/or control activation and differentiation of T cells outside the Bcell follicle. Here, we present the first specific assays for TFR cellfunction in vitro and show that sorted wild type TFR cells from thelymph node are extremely potent at inhibiting antibody production, butnot very effective at suppressing activation of naïve T cells.PD-1^(−/−) TFR cells inhibit naïve T cell activation and attenuateantibody production in vitro to a greater extent than WT TFR cells. Ourstudies also demonstrate the dynamic control of antibody production bylymph node TFH and TFR cells. Initially, our attempts to activate Bcells in vitro with total CD4⁺CXCR5⁺ cells resulted in little IgGsecretion. However, when we separated TFH cells from TFR cells and usedthese TFH cells in such experiments, we could detect robust IgGproduction. Of note, during an immune response to peptide/CFA, the invivo dLN TFR:TFH ratio is ˜1:5. When we cultured TFR and TFH cells atthis ratio, little antibody production was observed.

TFR cells tend to be present predominantly at the borders of germinalcenters, which may be explained by their relatively lower expression ofCXCR5 compared to TFH cells, though other chemokines also may haveroles. It is possible that close proximity of TFR cells to germinalcenter borders enables them to interact with TFH cells as they enter.This could make TFR cells the “gate-keepers” of the germinal center,inhibiting TFH cells as they enter and gain access to B cells undergoingsomatic hypermutation and class switch recombination. Furthermore, ourstudies suggest that the balance between TFR and TFH cells within thegerminal center itself may modulate the type and extent of humoralresponses. The relative roles of TFR and TFH cells also may depend onthe source or strength of antigenic stimulus, cytokine milieu, andtissue microenvironment, and further work is needed to investigate theseissues.

Surprisingly, we found substantial populations of TFR cells in the bloodof mice. There are a number of reports describing TFH cells in thecirculation of humans^(6, 7) and one in mice⁹. To our knowledge, ourwork is the first description of TFR cells in the blood of any organism.In humans, blood TFH cells have been shown to provide B cell help forthe production of antibody in vitro. Some studies show more efficient Bcell antibody production by blood TFH cells compared to blood CXCR5⁻cells^(6, 7) whereas other studies find no differences between blood TFHand CXCR5⁻ cells³⁰. Differences might relate to mixtures of blood TFHand TFR cells and their relative ratios in these experiments. Since mostwork describing blood TFH cells was done in humans, little is knownabout the requirements for blood TFH differentiation and function. Herewe show that murine blood TFH and TFR cell generation requires signalsthrough ICOS and CD28, two costimulatory receptors essential forcontrolling TFH cells in the lymph node. Previous work showed that CD28is essential for TFR cells in lymph nodes²¹. Here we demonstrate lymphnode TFR cell generation also requires ICOS signaling.

Our transfer studies show that blood TFR cells are functional and canregulate antibody production in vivo. To study TFH and TFR function, wetransferred blood TFH cells alone or with TFR cells into CD28^(−/−) orTCRα^(−/−) mice, which lack both blood and lymph node TFH and TFR cells.This approach allowed us to analyze TFH and TFR cell function separatelyfrom differentiation. Our transfer studies demonstrate effective andspecific control of humoral responses by blood TFH and TFR cells. BloodTFR cells are extremely potent at inhibiting TFH cell mediated antibodyproduction, even when relatively few cells are transferred. Wehypothesize that blood TFR cells may represent a central memory poolthat can be utilized to modulate humoral immunity, analogous to recentlyreported FoxP3⁺ cells with regulatory memory to self-antigens³¹ andsimilar to a proposed role for blood TFH cells⁷. High expression ofCD62L and CD44 on blood TFR cells along with their ability to home backto lymph nodes strongly support this idea. Blood TFH cells may migrateto lymph nodes and interact with cognate B cells rapidly upon antigenexposure, whereas naïve T cells need at least two to four days todifferentiate and upregulate CXCR5. Additionally, blood TFR cells homingto lymph nodes would be able to suppress early B cell responses, beforedLN nTregs could fully differentiate into TFR cells.

Beyond their ability to directly suppress antibody responses, TFR cellsmay be instrumental in determining B cell fates and control whether animmune response generates long-lived plasma cells or memory B cells. Forexample, cytokines produced by TFR cells may direct GC B celldifferentiation into plasma cells versus memory B cells. PD-1 and PD-1ligand deficiency result in decreased numbers of long-lived plasmacells¹⁶, and further work is needed to determine if this is related tothe enhanced PD-1^(−/−) TFR cell numbers and suppressive capacity. IfTFR cells can direct B cell fates, this would have implications forrational design of vaccines. We have also identified some of thepotential relative roles of TFH and TFR cells in autoimmunity. Forexample, PD-1 deficiency on autoimmune-prone backgrounds acceleratesdisease pathologies. It is possible that autoimmune-prone backgroundsmay lead to inhibition of TFR differentiation and function. Thisinformation will be important for therapeutic strategies using TFRcells. By expanding either TFH or TFR cells from patient blood in vitro,it may be possible to enhance antibody responses by transferring TFHcells or to inhibit systemic autoimmunity by transferring TFR cells.

In summary, we define a new role for PD-1 in regulating immuneresponses, by inhibiting differentiation and function of T follicularregulatory cells in both lymph node and blood. Our research has provideda better understanding of TFR and TFH interactions, and has thepotential to provide novel insights into mechanisms that regulatehumoral immunity and applications of those mechanisms in, for example, avaccination regimen. Our understanding of how PD-1 regulates humoralimmunity suggests suggest strategies for manipulating this pathway toenhance protective immunity and long-term memory or to inhibit systemicautoimmunity.

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Example 2. T Follicular Regulatory Cell (TFR) Cellular TherapyStrategies

1.) Enrichment and Injection of TFR Cells from Blood.

In order to skew the TFH:TFR balance, TFR cells will be purified fromperipheral blood. First, lymphocytes from peripheral blood will beisolated by sucrose density centrifugation and stained withfluorescently labeled antibodies. TFR cells will be purified andseparated from TFH cells by flow cytometry. TFR cells will be sorted viaflow cytometry based on surface markers of CD4⁺CXCR5⁺ICOS⁺GITR⁺ and thenadministered intravenously to patients. These patients include a)individuals with autoimmune diseases with pathogenic antibodies (such assystemic lupus erythematosis) as a means to inhibit immunopathologyrelated to these pathogenic antibodies, b) individuals undergoingtransplantation (to prevent pathogenic effects of antibodies duringtransplant rejection), c) patients undergoing gene therapy or stem celltherapy to prevent development of antibodies that would abrogate theintended beneficial effects of the transferred gene or cell.

2.) Enrichment/Activation and Injection of TFR Cells from Blood.

In order to skew the TFH:TFR balance and to promote heightenedsuppressive function of TFR cells, TFR cells will be sorted andactivated in vitro prior to administration into patients. Lymphocytesfrom peripheral blood will be isolated by sucrose density centrifugationand stained with fluorescently labeled antibodies. TFR cells will besorted via flow cytometry based on surface markers ofCD4⁺CXCR5⁺ICOS⁺GITR⁺ and activated in vitro by using a soluble anti-CD3antibody along with autologous CD19+ B cells from blood to trigger TCRsignaling and activation. After activation, TFR cells will be given topatients intravenously.

3.) Enhanced Activation of TFR Cells by Blocking PD-1 Pathway.

In order to skew the TFH:TFR balance and to promote heightenedsuppressive function of TFR cells, TFR cells will be sorted andactivated in vitro in the presence of PD-1 or PD-L1 antagonists, whichshould enhance TFR cell numbers and suppressive capacity. Lymphocytesfrom peripheral blood will be isolated by sucrose density centrifugationand stained with fluorescently labeled antibodies. TFR cells will besorted via flow cytometry based on surface markers ofCD4⁺CXCR5⁻ICOS⁺GITR⁺ and activated in vitro by using a soluble anti-CD3antibody along with autologous CD19+ B cells from blood to trigger TCRsignaling. PD-1 antagonists (monoclonal antibodies and/or small moleculeinhibitors) are added during activation to enhance activation of TFRcells. After activation, TFR cells are administered to patientsintravenously.

4.) Enrichment and Injection of Tfr Cells to Induce Long Term MemoryDuring Vaccination.

The goal of this strategy is to skew TFH:TFR balance in the blood duringB cell memory development and preferentially promote development of longlived B cell memory cells by inhibiting plasma cell differentiation. TFRcells will be sorted via flow cytometry and injected intravenously intoindividuals at the time of vaccination. Lymphocytes from peripheralblood will be isolated by sucrose density centrifugation and stainedwith fluorescently labeled antibodies. TFR cells will be sorted via flowcytometry based on surface markers of CD4⁺CXCR5⁺ICOS⁺GITR⁺ andreinjected intravenously.

5.) Induction of TFR Cells from nTreg Precursors by Blocking PD-1Pathway in Vitro.

In order to generate de novo TFR cells from Treg progenitors, naïveTregs are sorted from human blood by CD4+CXCR5−GITR+(or CD25+). Thesecells are activated with autologous CD19+ B cells with anti-CD3. Duringthis activation, PD-1 antagonists (blocking antibodies or small moleculeinhibitors) are used to block the PD-1 pathway. TFR cells are thensorted by flow cytometry as CD4+CXCR5+ICOS+GITR+ or and injected intopatients.

Example 3. Summary

T Follicular Regulatory (TFR) cells inhibit whereas T Follicular Helper(TFH) cells stimulate antibody production. Both TFR and TFH cells arefound in blood, however relatively little is known about thedevelopmental signals for these cells or their functions. Here wedemonstrate a new mechanism of control of antibody responses mediated byTFR and TFH cells. We find that circulating TFH and TFR cells aredistinct from lymph node effector TFR/TFH cells, and representmemory-like cells that have specialized function to patrol diverse sitesof potential antigen encounter. Circulating TFH cells can be potentlyactivated by DCs, home to germinal centers and produce large amounts ofcytokines. Although memory-like circulating TFR cells can be potentlyactivated in lymph nodes, they have a lower capacity to suppress B cellresponses than lymph node TFR cells. Therefore, memory-like circulatingTFH cells are able to provide quick and robust systemic B cell help.

Follicular helper T (TFH) cells are a subset of CD4⁺ T cells thatstimulate and maintain the germinal center reaction, enabling B cells toproduce high affinity antibodies. TFH cells are defined by CXCR5, thechemokine receptor which directs them to the B cell zone via gradientsof the chemokine CXCL13 (Breitfeld et al., 2000; Crotty, 2011). TFHcells express the transcription factor Bcl6 which facilitates CXCR5expression, as well as production of IL-21, a key cytokine that helps Bcells to undergo affinity maturation and produce antibody (Johnston etal., 2009; Nurieva et al., 2008; Nurieva et al., 2009). In addition, TFHcells can produce other cytokines including IFNγ IL-17 and IL-4 that arethought to influence the germinal center reaction by helping withselection of antibody isotypes during class switch recombination.Significant plasticity in cytokine production exists within in the TFHlineage, so distinguishing TFH from Th1, Th2 and Th17 cells can bechallenging (Cannons et al.). Lymph node (LN) TFH differentiation isthought to be a two step process: T cells are initially primed bydendritic cells and subsequently interact with B cells in the germinalcenter, culminating in full effector potential (Crotty, 2011; Goenka etal., 2011; Haynes et al., 2007; Poholek et al., 2010).

Follicular regulatory T (TFR) cells are a newly defined population ofCXCR5⁺CD4 T cells that until recently were hidden within TFH gatingstrategies. Like TFH cells, TFR cells express high levels of CXCR5, thecostimulatory molecule ICOS, and the coinhibitory molecule PD-1 (Chunget al., 2011; Linterman et al., 2011; Sage et al., 2013; Wollenberg etal., 2011). Importantly, TFR cells are thought to originate from thymicTreg (tTreg) precursors, in contrast to TFH cells which develop fromnaïve T cells (Chung et al., 2011; Sage et al., 2013).

TFR and TFH cells have diametrically opposing roles in regulatinghumoral immune responses; TFH cells stimulate humoral immunity, whereasTFR cells potently suppress humoral immune responses (Chung et al.,2011; Linterman et al., 2011; Sage et al., 2013; Wollenberg et al.,2011). The mechanisms by which TFR cells suppress the germinal centerreaction are still unclear. It is not known whether TFR cells suppressTFH cells, germinal center B cells, or both. Understanding how TFR cellsinhibit humoral immunity has the potential to enable improvedvaccination strategies.

TFH and TFR cells are not only present in LNs, but also in thecirculation. Circulating TFH cells from humans can provide help to Bcells in vitro (Morita et al., 2011; Schaerli et al., 2000), andcirculating TFH cells from mice can potently stimulate B cells in vivo(Sage et al., 2013). A subset of human blood TFH cells with CXCR5expression comparable to lymph node TFH cells but lower levels of PD-1and ICOS expression has been postulated to represent memory cells(Breitfeld et al., 2000; Morita et al., 2011; Rasheed et al., 2006).Therefore, it is possible that circulating TFH cells may give rise tomemory TFH cells (Fazilleau et al., 2007; Hale et al., 2013; Sage etal., 2013). Although there is evidence for the presence of memory TFHcells, the origin and function of these cells is not yet clear(Fazilleau et al., 2007; Hale et al., 2013; Luthje et al.; MacLeod etal., 2011; Marshall et al., 2011). If blood TFH and TFR cells possessproperties of memory cells, then they could serve as attractivecandidates for immune monitoring and cellular therapy (Morita et al.,2011; Sage et al., 2013; Saito et al., 2005; Schaerli et al., 2000;Simpson et al., 2010).

Elucidating the relationships between lymph node TFH and TFR cells andcirculating TFH and TFR cells may provide insights into TFH (and TFR)memory cell development and function (Crotty, 2011). Although lymph nodeTFR and TFH cells depend on CD28, ICOS and B cells for development, thespecific cues for blood TFH and TFR cell development and maintenance arenot yet clear. Circulating TFH cells in humans appear to differ fromlymph node TFH cells by microarray analysis, however these differencesmay be due to decreased activation in the blood or contaminating TFRcells (Rasheed et al., 2006). The most straightforward explanation forthe presence of TFH and TFR cells in the circulation is that some TFHand TFR cells from the germinal center “decide” to leave the lymph node.If this hypothesis is true, then circulating TFH and TFR cells wouldrequire lymph node TFH and TFR cells for their development. In supportof this hypothesis, TFH and TFR cells are completely missing from thelymph nodes and blood of CD28 and ICOS deficient mice (Bauquet et al.,2009; Bossaller et al., 2006; Sage et al., 2013). However, there aredata that are inconsistent with this hypothesis. PD-1 deficient micehave increased TFH cells in the blood, but not in the lymph node (Sageet al., 2013). In addition, lymph node TFH and TFR cells develop withsimilar kinetics as blood TFH and TFR cells (Sage et al., 2013).Moreover, recent tracking experiments suggest that GC TFH cells areunable to gain access to the circulation (Shulman et al., 2013). Thesefindings suggest that circulating and lymph node TFH and TFR cells maydevelop independently.

Here we demonstrate that “memory-like” blood TFR and TFH cells aredistinct and specialized cellular subsets that potently and systemicallycontrol antibody production. Memory-like blood TFH and TFR populationsdiffer from lymph node TFR and TFH “effector” cells in several ways.Memory-like blood TFH and TFR cells can circulate throughout the bodypatrolling for antigen, in contrast to LN TFH and TFR cells. Afterhoming to lymph nodes, blood TFH cells require repriming with dendriticcells to be completely functional; these activated TFH cells produceenhanced amounts of cytokines and dominate the CXCR5 population comparedto lymph node TFH cells. In addition, we find that blood TFR cells havedecreased suppressive capacity in vitro, compared to lymph node TFRcells. Since blood TFH cells are more potently activated and blood TFRcells are less suppressive, the blood CD4⁺CXCR5⁺ population as a wholeis poised to form a quick memory-like response throughout the bodywherever re-exposure to antigen may occur. Taken together, these studiesidentify a novel mechanism by which circulating TFH cells control B cellresponses in vivo.

Results Differentiation of Circulating TFH and TFR Cells Require Primingby Dendritic Cells

We recently demonstrated that T follicular regulatory (TFR) and Tfollicular helper (TFH) cells are present in the blood of mice. Todetermine if blood and lymph node TFH and TFR cells have phenotypicdifferences, we first compared the expression of CXCR5 and other cellsurface markers on murine TFH and TFR cells from lymph node and blood.Initially, we analyzed CXCR5 and ICOS expression on circulating andlymph node cells 7 days after subcutaneous immunization with NP-OVA inCFA, an immunization that causes robust differentiation of both TFH andTFR cells (Sage et al., 2013). TFH cells, defined asCD4⁺ICOS⁺CXCR5⁺FoxP3⁻CD19⁻, greatly expanded in the draining lymph nodes(dLN) and blood of immunized mice compared to unimmunized controls (FIG.15A-B). The blood TFH cells had lower, but still substantial, expressionof CXCR5 (FIG. 22A-FIG. 22E). Expression of the essential TFHcostimulatory molecule ICOS was much lower on blood TFH compared to LNTFH cells (FIG. 22A-FIG. 22E). Intracellular staining of the cell cyclemarker Ki67 revealed that fewer blood TFH cells were in cell cyclecompared to dLN TFH cells. However, blood TFH cells had much higher Ki67expression than the total CD4 T cell population (FIG. 22E).

TFR cells, defined as CD4⁺ICOS⁺CXCR5⁺FoxP3⁺CD19⁻ cells, differentiatedsignificantly in both the dLN and blood after immunization (FIG.22F-FIG. 22G). Draining LN TFR cells expressed lower levels of CXCR5than dLN TFH cells. Blood TFR cells expressed even lower levels of CXCR5than dLN TFR cells (FIG. 22H). ICOS expression was also greatlyattenuated in blood TFR cells compared to dLN TFR cells (FIG. 22I).However, Ki67 intracellular staining revealed that there were similarproportions of dLN and blood TFR cells in cell cycle (FIG. 22J). Takentogether, these data indicate that circulating TFH and TFR cells arephenotypically distinct from dLN TFH and TFR cells and express lessCXCR5 and ICOS.

Next we analyzed the developmental cues necessary for circulating TFHand TFR cells. LN TFH cells depend on dendritic cells for initialdifferentiation (Goenka et al.) and require B cells for maintenance ofthe TFH phenotype, as well as optimal CXCR5 and ICOS expression (Choi etal., 2011; Kerfoot et al., 2011; Poholek et al., 2010). LN TFR cellsalso depend on B cells for differentiation (Linterman et al., 2011). Todetermine if dendritic cells are necessary for the generation ofcirculating TFH and TFR cells, we utilized CD11c DTR bone marrowchimeric mice to deplete dendritic cells. We used bone marrow chimericmice for these in vivo studies to circumvent the lethality related tostromal expression of CD11c in the brain. We administered diphtheriatoxin every 2 days starting on day 0 during an NP-OVA immunization, andcompared the generation of blood and dLN TFH and TFR cells 7 days postimmunization. Diphtheria toxin administration resulted in depletion ofmost, but not all, dendritic cells in the dLN of CD11cDTR mice (FIG.15A). Depletion of dendritic cells led to significantly lowerpercentages of Ki67⁺ TFH and TFR cells in both the dLN and blood,suggesting that dendritic cells are key for differentiation of both dLNand blood TFH and TFR cells (FIG. 15B).

Next, we investigated whether antigen presentation by dendritic cellswas sufficient for development of circulating TFH and TFR cells bycomparing antigen presentation by WT and MHC II deficient (Ciita^(−/−))dendritic cells using adoptive transfer approaches. We differentiated WTor Ciita^(−/−) bone marrow derived dendritic cells (BMDCs) with GM-CSF,activated them with LPS and pulsed them overnight with NP-OVA. Afterwashing the cells thoroughly, we adoptively transferred these Ag-pulsedBMDCs subcutaneously into WT mice, and analyzed the dLN and blood forTFH and TFR cell development 5 days later. When NP-OVA-pulsed WT BMDCswere adoptively transferred, there were substantial increases inpercentages of total CD4+CXCR5+ cells in both the dLN and blood (FIG.15C). When TFH cells were quantified, we found that transfer ofantigen-pulsed WT BMDCs caused a significant increase in the proportionsof TFH cells in both the lymph node and blood (FIG. 15D). In contrast,adoptive transfer of NP-OVA-pulsed MHC II-deficient BMDCs resulted inmodest increases in percentages of CXCR5⁺ cells in the dLN and blood(FIG. 15C-D). Antigen-pulsed WT BMDCs also stimulated a significantincrease in the proportion of TFR cells in the lymph node. However,Ag-pulsed WT BMDC did not result in increased blood TFR cells,suggesting that cues for blood TFR cell generation can be supplied bysubcutaneous immunization but not DC transfer approaches.

Since B cells are required for full differentiation of dLN TFH and TFRcells, we next tested whether B cells are required for the generation ofcirculating TFH and TFR cells. We immunized WT and “μMT” mice (that lackmature B cells due to the lack of surface IgM expression) with NP-OVA,and compared dLN and circulating TFH and TFR cells. Similar to previousreports, the percentages of TFH cells and TFR cells in the dLN wereseverely attenuated in μMT mice compared to WT mice (FIG. 15E-F)(Poholek et al., 2010). Surprisingly, the percentages of TFH and TFRcells within the blood of μMT mice were equivalent to those of WT mice,suggesting that blood TFH and TFR cells require distinct cues fordevelopment compared to LN TFH and TFR cells. Furthermore, since bloodTFH and TFR cell numbers did not increase in the μMT mice compared to WTmice, it is unlikely that the absence of dLN TFH and TFR cells is due tomigration of these cells into the blood. Since the lack of mature Bcells attenuated TFH and TFR numbers in the dLN but not the blood, theμMT mice have a blood:LN TFH ratio 6 times higher than that of WT miceand a blood:LN TFR ratio 2 fold higher than WT mice (FIG. 15G). Takentogether, these data indicate that circulating TFH and TFR cells requiresignals from DCs for their generation.

Blood TFH and TFR Cells Exit the Lymph Node Via SW Signals

To further understand the relationship between LN and blood TFH and TFRcells, we next investigated the cues responsible for exit of TFH and TFRcells from the dLN and entry into the circulation.Sphingosine-1-phosphate (S1P) levels control T cell exit from the lymphnode into the efferent lymph (Matloubian et al., 2004). High levels ofS1P in blood and lymph act as a chemoattractant for T cells. Thisdirected migration can be blocked by expression of CD69 on newlyactivated cells, which downregulates S1P receptors (Cyster and Schwab,2011; Shiow et al., 2006). Based on these data, we hypothesized thatblood TFH and TFR cells would have low CD69 expression. To test thishypothesis, we compared CD69 expression on TFH and TFR cells from thedLN and blood of mice that were immunized with NP-OVA 7 days previously.Blood TFH and TFR cells had ˜2-3 fold lower CD69 expression compared toTFH and TFR cells from the lymph node (FIG. 16A-B).

To test whether TFH and TFR cells utilize S1P signals to exit the lymphnode via the efferent lymphatic system, we used FTY720 which preventscells from responding to S1P gradients and prevents lymphocyte egressfrom lymphoid tissues (Cyster, 2005; Cyster and Schwab, 2011). Weimmunized mice with NP-OVA and then administered FTY720 to mice every 2days (starting on day 2) and harvested organs 7 days after NP-OVAimmunization. Blood TFH cells were virtually absent in mice thatreceived the FTY720 treatment (FIG. 16C). TFR cells showed a similardependence on S1P signals and very few were present in the blood afterFTY720 administration (FIG. 16D). However, the numbers of dLN TFH andTFR cells were not significantly changed by FTY720 treatment. Toinvestigate the kinetics of blood TFH and TFR circulation, we immunizedmice with NP-OVA and administered FTY720 on day 7 after immunization andanalyzed TFH and TFR cells after 3 or 6 hours after FTY720administration. When we treated mice with FTY720 only for 3 or 6 hoursprior to analysis, there was also a marked attenuation of both TFH andTFR numbers, demonstrating TFH and TFR cells exit the blood quickly,within only a few hours (FIG. 16E).

To analyze TFH and TFR cell exit from the dLN and entry into efferentlymph, we conducted experiments in which the efferent lymph wascollected via a thoracic duct cannulation method (Massberg et al.,2007). We detected robust percentages of both TFH and TFR cells in thelymph (FIG. 16F-G). The percentages of TFH cells in lymph and dLN weresimilar and the percentages of TFR cells in lymph and blood were similar(FIG. 16F-G). Lymph TFH and TFR cells had lower levels of ICOS comparedto dLN TFH and TFR cells, but levels similar to blood TFH and TFR cells(FIG. 16H). Thus, both TFH and TFR cells have low ICOS expression uponexiting the dLN and entering the lymph, but do not further downregulateICOS after entry into the circulation. Taken together, these dataindicate that both TFH and TFR cells use S1P signals to leave the dLN,and their circulation in the blood is transient, on the order of hours.

Circulating TFH and TFR Cells Migrate to Diverse Lymph Nodes and Tissues

To test the hypothesis that circulating TFH and TFR cells represent amemory pool of cells that can migrate to diverse secondary lymphoidorgans where they can be reactivated to become effector cells, we nextinvestigated blood TFH and TFR cell trafficking in vivo. To monitorblood TFH and TFR cell behavior in vivo we crossed Actin-CFP mice withFoxP3-IRES-GFP mice to create mice that report FoxP3 expression but canalso be tracked in vivo by CFP expression (these mice are referred to asActin^(CFP)-FoxP3^(GFP) mice). We immunized 20 Actin^(CFP)-FoxP3^(GFP)mice with NP-OVA and 7 days later sorted total CD4⁺ICOS⁺CXCR5⁺CD19⁻cells to keep blood TFH and TFR cells in endogenous proportions. Wetransferred these cells into CD28^(−/−) recipients, which cannotgenerate dLN or blood TFH and TFR cells. Transfer into CD28^(−/−)recipients allowed us to transfer very small numbers (2×10⁴) of cells,yet avoid homeostatic proliferation since CD28^(−/−) mice havesubstantial T cell populations (Sage et al., 2013). We immunizedCD28^(−/−) recipients with NP-OVA subcutaneously and 7 days laterisolated lymphoid organs and other tissues and analyzed transferredcells. We found significant populations of transferred cells in alllymph nodes tested, demonstrating that blood TFH and TFR cells havehoming properties similar to central memory cells (FIG. 17A).Surprisingly, more than half of the CD4 T cells in the skin around theimmunization site were transferred blood CXCR5⁺ cells, demonstratingthat circulating TFH and TFR cells can home to peripheral tissues aswell as lymphoid organs. When we examined the transferred cells for ICOSand CXCR5 expression we found that the transferred cells in the lymphnodes and skin highly upregulated ICOS surface expression. However, ICOSexpression remained low in the blood and spleen, similar to levels ofICOS expression on sorted blood CXCR5⁺ cells prior to transfer (FIG.11A-FIG. 11C, 17A). In comparison, CD28^(−/−) recipient CD4 T cells hadvery low ICOS expression. CXCR5 expression was maintained on about 40percent of transferred cells in the draining (inguinal) lymph node.Other organs, particularly the blood, showed lower CXCR5 expression.

To determine specifically where TFH and TFR cells homed, we usedFoxP3^(GFP) to identify TFR cells in the CFP⁺ transferred population.The FoxP3⁻ TFH cells predominated in the transferred population (FIG.17C). Compared to the percentage of TFR cells in the input population,the percentage of TFR cells in the CXCR5⁺CFP⁺ gate in the variouslymphoid organs was much lower (FIG. 17D). Therefore, blood TFR cellsare more short-lived or proliferate less after homing to various tissuesthan blood TFH cells. The highest proportion of TFR to TFH cells in therecipients was in the mesenteric lymph node. The overall reduction inTFR cells was not due to CXCR5 downregulation on TFR cells, since thisdecrease was also seen when FoxP3⁺ cells were gated on the total CFPtransferred population (FIG. 17E).

To determine if blood TFH and TFR cells are capable of migrating to theB cell zone in order to perform effector functions after homing to lymphnodes, we analyzed the location of transferred cells in dLNs ofrecipient mice. We immunized CD45.2 WT mice with NP-OVA, transferredsorted blood TFH cells (CD4+ICOS+CXCR5+GITR−CD19−, a gating strategy wehave established previously (Sage et al., 2013)) to CD45.1 recipientsthat were subsequently immunized with NP-OVA. 7 days later we foundCD45.2+ blood TFH cells interacting with IgG1+ class switched B cellsboth within germinal centers and outside germinal centers in theinterfollicular zone of the dLN of recipient mice (FIG. 17F). Likewise,when we transferred blood TFR cells (CD4+ICOS+CXCR5+GITR+CD19−) intorecipient mice that were subsequently immunized, we found transferredblood TFR cells interacting with IgG1⁺ class switched B cells in theinterfollicular zone, and sometimes directly in the germinal centers ofthe dLN of recipient mice (FIG. 17G). Taken together, these dataindicate the circulating TFH and TFR cells are capable of homing tonumerous secondary lymphoid organs and tissues to perform effectorfunctions.

Circulating TFH and TFR Cells are More Potently Activated after TransferIn Vivo

Next we compared the function of circulating TFH and TFR cells with LNTFH and TFR cells. We hypothesized that if blood TFH and TFR cells havecharacteristics of memory cells, they would have increased ability to beactivated in vivo compared to dLN TFH and TFR cells. To investigate thisissue, we immunized Actin^(CFP)-FoxP3^(GFP) mice with NP-OVA and 7 dayslater sorted total CD4⁺CXCR5⁺CD19⁻ cells from the dLN and blood andadoptively transferred these cells intravenously into CD28^(−/−)recipients, which were immunized with NP OVA. As in FIG. 18A, we couldreadily detect small populations of transferred cells in the dLN ofrecipients 7 days later based on the presence of CFP (FIG. 18A).

Since Bcl6 expression is essential for TFH cell function, weinvestigated if blood TFH cells could upregulate Bcl6 after entering andbeing activated in the dLN. We found slightly higher Bcl6 expression intransferred blood TFH cells compared to transferred dLN cells (FIG.18B). These findings indicate that circulating TFH cells can becomeeffector TFH cells upon homing back to a lymph node during an immuneresponse. Next we compared ICOS expression on transferred dLN and bloodcells (“CXCR5 Transfer”) in the dLN or blood (the “assayed population”)of immunized CD28^(−/−) recipients. We also compared ICOS expression ondLN and blood TFH and TFR cells from immunized WT mice (“endogenous WT”)in order to facilitate comparisons. ICOS is expressed at a very lowlevel on endogenous WT blood TFH cells from WT mice (FIG. 18C). ICOSexpression was dramatically increased on blood TFH cells after homingback to the lymph node (dLN TFH assayed population, blood CXCR5transfer) compared both to endogenous WT blood TFH cells and even dLNtransferred TFH cells (dLN TFH assayed population, dLN CXCR5 transfer)(FIG. 18C). The increased ICOS expression on circulating TFH cells wasnot unique to the TFH subset, because blood TFR cells that were found inthe dLN also greatly upregulated ICOS expression compared to endogenousblood TFR cells and transferred LN TFR cells (FIG. 18D). CXCR5expression was similar in blood TFH cells upon rehoming to the lymphnode and transferred lymph node TFH and TFR cells in the lymph node(FIG. 18E). Blood TFR cells had slightly lower CXCR5 upon rehoming tothe lymph node compared to transferred LN TFR cells in the LN (FIG.18F).

To determine if the increase in Bcl6 and ICOS translated into anincrease in effector cytokine production by the circulating TFH cells,we compared cytokines produced by the transferred dLN or blood TFH cellsfound in the dLN after immunization. We found no difference in the verylow, but detectable, intracellular IL-21 expression in the transferredblood TFH cells and transferred dLN TFH cells. In contrast, there was asubstantial increase in intracellular IL-17A produced by transferredcirculating TFH cells compared to transferred dLN TFH cells (FIG. 18G).Intracellular IL-2 expression was also ˜2 fold higher in transferredcirculating TFH cells, compared to transferred dLN TFH cells (FIG. 18G).Taken together, these data indicate that circulating TFH cells (and TFRcells) can be potently re-activated upon homing to secondary lymphoidorgans, consistent with a memory phenotype.

Circulating TFH Cells Require Dendritic Cells for Restimulation and haveMemory Properties

Next we investigated the signals required for reactivation of blood TFHcells. We determined if dendritic cells and/or B cells were necessaryfor reactivation of circulating TFH cells using in vitro systems tosensitively evaluate activation and effector functions of TFH cells. Weimmunized 20 WT mice with NP-OVA subcutaneously and 7 days laterpurified dLN and circulating TFH cells by sortingCD4⁺ICOS⁺CXCR5⁺GITR⁻CD19⁻ cells, as we previously reported (Sage et al.,2013)). We compared the reactivation of these TFH cells followingculture with B cells or dendritic cells (also sorted from dLN ofimmunized mice) in the presence of anti-CD3 and anti-IgM. We evaluatedBcl6 and Ki67 expression to compare the activation and effectorpotential of dLN and circulating TFH cells. When cultured with B cells,dLN TFH cells had a higher percentage of Bcl6⁺Ki67⁺ cells thancirculating TFH cells (FIG. 19A-B). Culturing dLN TFH cells with DCsonly modestly increased the percentage of Bcl6⁺Ki67⁺ cells, as comparedto culture with B cells. In contrast, culture of circulating TFH cellswith DCs led to a greatly enhanced percentage of Bcl6⁺Ki67⁺ cells, ascompared to circulating TFH cells cultured with B cells or lymph nodeTFH cells cultured with DCs (FIG. 19B). We next investigated if theincreased Bcl6 expression in the circulating TFH cells cultured with DCswas associated with enhanced cytokine production. We found profoundincreases in intracellular IFNγ as well as smaller increases in IL-21,IL-17A, IL-2 and IL-4 in circulating TFH cells compared to lymph nodeTFH cells cultured with DCs (FIG. 19C).

To determine if blood TFH cells require DCs for persistence in vivo, weadoptively transferred 1.5×10⁴ blood CD4⁺CXCR5⁺CD19⁻ cells fromimmunized Actin^(CFP)-FoxP3^(GFP) mice into CD11cDTR bone marrowchimeric mice that were immunized with NP-OVA. We administereddiphtheria toxin (DT) on days 0, 3 and 5 during a 7 day immunization todeplete DCs and measured the number of transferred cells 7 days later.We found that DC depletion resulted in fewer CFP⁺ cells persisting, aswell as a lower percentage of cells expressing CXCR5 on the transferredcells (FIG. 19D). Taken together, these data indicate that circulatingTFH cells need to be restimulated by dendritic cells and these TFH cellshave a superior ability to produce TFH cytokines.

We next determined if blood TFH and TFR cells could persist in vivosimilarly to memory T cells and dominate a GC reaction upon re-exposureto antigen by conducting parabiosis experiments. Actin^(CFP)-Fox^(GFP)mice were immunized with NP-OVA and the circulatory systems of thesemice were surgically joined to WT mice, allowing transfer of blood TFHand TFR cells to the adjoining mouse (FIG. 19E). After being joined for19 days, chimerism was confirmed, and mice were separated. The WT“non-immunized” mouse was then immunized with NP-OVA and 7 days laterorgans were harvested and analyzed. We compared the percent of CFP+cells in the naïve as well as the CXCR5+ICOS+ populations. The CFP+population in the naïve gate (from the non-draining lymph node) isindicative of baseline chimerism, and contributions above thispercentage in the CXCR5+ICOS+ gate are indicative of increased bloodTFH/TFR cells in the germinal center. We found significantly enhancedCFP+ cells in the CXCR5+ICOS+ gate in the draining lymph node and lessso in the non-draining (cervical) lymph node (FIG. 19E). We also foundsubstantial increases in the CFP population in the spleen, blood and(more dramatically) in the skin. Together these data demonstrate thatthe blood memory-like CXCR5+ population can out compete and dominate theGC reaction upon re-exposure to antigen.

Next we used adoptive transfer approaches to analyze TFH and TFR cellsat memory time points. We transferred total blood CXCR5+ cells fromNP-OVA immunized Actin^(CFP)-Fox^(GFP) mice to WT mice. After 30 days,WT recipients were immunized with NP-OVA and draining lymph nodes wereharvested 7 days later. We found a small, but substantial population ofCFP positive cells in the draining lymph node (FIG. 19F). Although themajority of these cells were TFH cells, there was a very smallpopulation of TFR cells that persisted. The TFH cells had high ICOSexpression and a sizeable population that maintained CXCR5 expression,suggesting functionality. Taken together these data indicate that bloodTFH and TFR cells need to be reactivated by DCs, can dominate the GCreaction upon exposure to antigen, and can persist in vivo similar tomemory cells.

TFR Cells Potently Suppress T Cell and B Cell Activation

Next we compared the suppressive functions of lymph node and blood Tfrcells. Since the mechanisms by which TFR cells exert their suppressiveeffects are not well understood, we first developed a series ofsensitive in vitro suppression assays for analyzing TFR cellsuppression. We immunized more than 20 WT mice with NP-OVAsubcutaneously and 7 days later sorted TFH (CD4⁺ICOS⁺CXCR5⁺GITR⁻CD19⁻)and TFR (CD4⁺ICOS⁺CXCR5⁺GITR⁺CD19⁻) cells from the dLN. We cultured TFHcells (with or without TFR cells) together with total B cells ordendritic cells (sorted from the dLN of immunized mice) for 6 days inthe presence of anti-IgM and anti-CD3. CXCR5 remained highly expressedon TFH cells whether cultured with B cells or DCs (FIG. 20A). Theaddition of TFR cells did not cause downregulation of CXCR5 on TFHcells, suggesting that TFR cells do not suppress TFH cells by divertingthem away from the germinal center by downregulation of chemokinereceptors (FIG. 20A). Bcl6 expression was highly expressed on the TFHcells when cultured with dendritic cells or B cells (FIG. 20B). However,Bcl6 expression in the TFH cells was attenuated profoundly in thepresence of TFR cells and DCs (but not B cells), suggesting that TFRcell repriming by DCs may increase TFR suppressive capacity. TFH Ki67expression also was strongly suppressed by TFR cells in both B cell andDC culture conditions, suggesting that a key TFR cell suppressionmechanism is inhibition of cell cycling in TFH cells (FIG. 20C).

To determine if TFH cytokine production was also suppressed by TFRcells, we analyzed TFH cells from in vitro cultures for cytokineproduction by intracellular staining. We found that TFR cells suppressedIFNg, IL-21, IL-10 and TNFα (FIG. 20D). TFR cells had no effect onIL-17A or IL-2 and caused an increase in IL-4. Thus, TFR cells areselective in the TFH cytokines that they suppress, at least in this invitro model. Together, these studies suggest that TFR cells exert theirsuppressive effects by inhibiting TFH cell proliferation and cytokineproduction.

We next investigated whether TFR cells also affect B cell function. Wecultured dLN TFH and B cells with or without TFR cells in the presenceof anti-CD3 and anti-IgM for 6 days and then analyzed the B cells fromthese cultures. The germinal center B cell marker GL7 was highlyupregulated on B cells when cultured with TFH cells, but markedlyreduced when TFR cells were present in co-cultures (FIG. 20E-F).Importantly, this in vitro suppression depends on the “TFR program” asFoxP3+CXCR5− cells sorted from unimmunized lymph nodes did not attenuatethe expression of GL7 on B cells in contrast to TFR cells (FIG. 20F).Furthermore, Ki67 expression was greatly attenuated in B cells whencocultured with TFH and TFR cells as compared to culture with TFH cellsalone, demonstrating potent suppression of B cell activation by TFRcells (FIG. 20G). TFR cells also inhibited IgG1 expression in B cells. Asubstantial portion of B cells class switched to IgG1 when cultured withTFH cells but this was abrogated in the presence of TFR cells (FIG.20H-I). B cell class switch recombination suppression was dependent onthe TFR program, since non-TFR Tregs could not suppress IgG1 levels on Bcells (FIG. 20I). Taken together these data indicate that lymph node TFRcells can suppress TFH cell activation, as well as B cell activation andantibody production in vitro.

Weaker B Cell Suppression by Blood TFR Cells

Finally, since blood TFH cells can be reactivated more potently than LNTFH cells, we compared the suppressive functions of blood TFR cells anddLN TFR cells. We compared the abilities of dLN TFR and blood TFR cellsto suppress dLN TFH cells and B cells using the assays described above.TFH cells promoted IgG1 and GL7 expression in B cells (FIG. 21A). BloodTFR cells were able to greatly suppress IgG1 expression in B cells, butwere less potent than dLN TFR cells. Additionally, blood TFR cells wereable to attenuate GL7 expression, but this was not as robust as theattenuation by dLN TFH cells (FIG. 21B). Thus, dLN TFR cells potentlysuppress B cell activation but blood TFR cells suppress B cell responsesto a somewhat lesser extent than dLN TFR cells. Additionally, a lowerpercentage of blood TFR cells expressed Ki67, compared to dLN TFR cells,regardless of culture with B cells or DCs (FIG. 21C). Therefore, unlikeblood TFH cells, blood TFR cells are not more potently activated by DCsthan dLN TFR cells.

Since blood TFH cells are more potent than LN TFH cells and blood TFRcells are less inhibitory than LN TFR cells in vitro, we next comparedthe effect of the total blood CXCR5 (TFH/TFR cells) population on B cellactivation in vivo compared to dLN CXCR5 cells. We sorted total CXCR5+cells from dLNs or blood of immunized Actin^(CFP) Fox^(GFP) mice (whichhad the same TFH/TFR ratios) and adoptively transferred these cells toCD28^(−/−) recipient mice that were immunized as in FIG. 18A. When weanalyzed the draining lymph node 10 days after adoptive transfer, wedetected a greater percentage of both GC B cells and plasma cells in themice that received blood CXCR5+ cells (FIG. 21D-E). Taken together,these data indicate that stimulatory blood TFH cells are poised toprovide a quick and robust memory-like response in secondary lymphoidorgans.

Discussion

In this study we have determined that “memory-like” blood TFH and TFRcells differ from “effector” dLN TFH and TFR cells because they providesystemic B cell control in secondary lymphoid organs and not just at thesite of differentiation. We demonstrate that blood TFH and TFR cellshave distinct phenotypes and require different cues for generationcompared to lymph node TFH and TFR cells. Additionally, we show thatcirculating TFH and TFR cells can be potently activated by DCs indiverse secondary lymphoid organs and tissues. Finally, beyond theirability to home to and patrol diverse sites of possible antigenencounter, we find that blood TFH and TFR cells may provide effectorfunctions differing from LN TFH and TFR cells upon reactivation.Therefore, blood TFH and TFR cells constitute distinct subsets thatshare many hallmarks of memory cells and the blood TFH cells can providea quick and robust response to stimulate humoral immunity.

We find that dendritic cell signals are important for blood TFH and TFRdevelopment, but TFH and TFR cell numbers are not altered in mice thatlack mature B cells. This contrasts with lymph node TFH and TFR cellsthat are lacking in μMT mice (Haynes et al., 2007; Johnston et al.,2009; Linterman et al., 2011; Poholek et al., 2010). However, it isimportant to note that the μMT mouse has an altered lymph node structureand blood TFH/TFR cell behavior may be a consequence of thesealterations. Nevertheless, our studies suggest that circulatingmemory-like TFH and TFR cells are not derived from lymph node TFH andTFR cells, but differentiate in parallel with lymph node TFH and TFRcells.

The signals that elicit dLN versus circulating memory-like TFH and TFRdifferentiation are not clear. Full T cell activation in general isthought to occur through multiple serial contacts with dendritic cellsleading to full activation (Mempel et al., 2004). We hypothesize thattwo fates emerge for follicular CD4 T cells during differentiation withDCs. Subsets of TFH and TFR cells that have lower CXCR5 expression andlow CD69 after priming exit the lymph node via S1P gradients and aredestined to become blood memory cells. TFH and TFR cells with higherCXCR5 and high CD69 after priming follow CXCL13 gradients and migrate tothe B cell zone, where full differentiation and maintenance of the TFHand TFR effector phenotype occurs. Therefore, within the “window” forTFH and TFR differentiation, subtle differences in TCR and/orcostimulation can push the fate of the cells towards an effector ormemory TFH/TFR cell phenotype. Importantly, when TFH and TFR cells enterthe efferent lymph, they already have a surface phenotype similar toblood TFH and TFR cells, suggesting that the circulating/memory TFH andTFR cell phenotype is acquired during differentiation and not afterentry into the circulation. These data also demonstrate that circulatingTFH and TFR cells do not originate from GC TFH cells. This is consistentwith a recent study which demonstrated that GC TFH cells can movebetween the GC and interfollicular regions, but do not enter thecirculation (Shulman et al., 2013).

Our studies with the S1P agonist FTY720, as well as our transfer andtracking experiments, suggest that TFH and TFR cells recirculate fromthe blood to secondary lymphoid organs and tissues rather quickly, onthe order of hours. We hypothesize that the function of circulating TFHcells is to probe various organs for antigen and provide specialized androbust B cell help during secondary antigen exposure or systemicinfections. Relatively lower expression of CXCR5 on circulating TFH andTFR cells may allow these cells to better patrol T cell zones for DCspresenting antigen, while LN TFH and TFR cells may get “stuck” in the Bcell follicle due to high CXCL13 levels present there. Circulating TFRcells may help to regulate circulating TFH responses to ensure thatantigen-specific B cell responses are generated, but limithyperactivation or antibody production in distal secondary lymphoidorgans.

We previously showed that blood TFH cells stimulate more IgG productionin vivo than dLN TFH cells (Sage et al., 2013). Here we demonstrate thatupon reactivation by dendritic cells (but not B cells), blood TFH cellsproduce more cytokines, particularly IFNγ compared to dLN TFH cells.Classical experiments have demonstrated that IFNγ can inhibit B cellclass switching from IgG1 to IgG2a, and recent studies have suggestedthat directed IFNγ production, presumably by TFH cells, has a similareffect (Reinhardt et al., 2009). However, in our experiments, blood TFHcells do not skew antibody isotypes away from IgG1 to any particularisotype in vivo or in vitro (data not shown). Although IFNγ is thecytokine that is most highly expressed by blood TFH cells compared todLN TFH cells, there are slight increases in other cytokines as well.This suggests that blood TFH cells have an increased effector functionupon restimulation by DCs and are not simply Th1-skewed TFH cells. Thedistinct cytokine profile of blood TFH cells may lead to specializeddifferentiation and/or function of germinal center B cells or plasmacells.

Far less is known about the signals necessary for the differentiationand function of TFR cells than TFH cells (Chung et al., 2011; Lintermanet al., 2011; Sage et al., 2013; Wollenberg et al., 2011). LN TFR celldevelopment follows many of the same cues as TFH cells, such as the Bcell requirement in the lymph node as well as CD28 and ICOScostimulation (Linterman et al., 2011; Sage et al., 2013). It has beensuggested that subsets of Treg cells (such as TFR cells) may use Thelper transcription factors that are not normally expressed by Tregs tohelp position themselves near T helper cells and/or to program T helperspecific suppression (Josefowicz et al., 2012). For example, adiposetissue-resident Tregs, similar to other cells present in adipose tissue,depend on PPAR-γ (Cipolletta et al., 2012). We previously showed thatthe TFR program is essential for suppression of antibody production asCXCR5″ Tregs could not suppress antibody production in vivo (Sage etal., 2013). The exact mechanisms of TFR cell suppression are not known(Josefowicz et al., 2012). Here we show that lymph node TFR cellspotently suppress activation of TFH cells, but do not suppress CXCR5 orBcl6 expression in TFH cells cultured with B cells. Draining LN TFRcells can also suppress IFNγ, IL21, IL10 and TNFα expression by TFHcells. Interestingly, dLN TFR cells were unable to suppress IL17A or IL2production in dLN TFH cells, at least in in vitro models. In addition,we find that dLN TFR cells potently suppress dLN TFH-mediated B cellactivation, class switch recombination and germinal center markers.Importantly, non-TFR FoxP3⁺ Tregs were not able to suppress GL7 or classswitching to IgG1. Since these in vitro assays do not depend on distinctT and B cell zones, the inability of non-TFR cells to suppress B cellssuggests a “TFR program” beyond CXCR5 expression that is essential forTFR cell suppressive functions.

It is not currently known if T regulatory cells can possessimmunological memory. One report showed that self-reactive Tregs canpossess some form of regulatory memory at least within the skin(Rosenblum et al., 2011). Here we demonstrate that memory-like blood TFRcells can home to diverse secondary lymphoid organs, including the skin.Within the skin TFR cells may inhibit effector T cells or providespecific suppression of memory-like TFH cells and antibody secretingcells (ASCs). It also is possible that TFR cells may suppress formationof ectopic follicles that can arise during certain inflammatoryconditions within the tissue (Peters et al.). We hypothesize that themain role of blood TFR cells is to provide systemic control of humoralimmunity during an immune response by migrating throughout the body toorgans that may be exposed to antigens. Blood TFR cells may function toensure proper TFH control and thereby limit excessive or inappropriate Bcell help. Additionally, blood TFR cells may prevent humoralautoimmunity by raising the threshold for B cell activation insituations where an abundance of proinflammatory cytokines might bypassthe need for TFH help.

Since blood TFR cells require distinct cues compared to lymph node TFRcells, it is likely that blood TFR cells may be programmed differentlythan lymph node TFR cells. We show here that both lymph node and bloodTFR cells are able to attenuate B cell activation but blood TFR cellsare less suppressive. This difference may be due to differentialprogramming during development. Further experiments are needed toinvestigate these issues. Importantly, the TFH:TFR ratio, which isimportant in controlling humoral immunity (Sage et al., 2013), isroughly similar between LN and blood TFH:TFR cells. Therefore,functional differences between blood and LN CXCR5⁺ cells can beattributed to cell intrinsic differences in the TFH and TFRcompartments. By interrogating these cells individually, we show thatblood TFH cells are more potently activated upon restimulation (comparedto LN TFH cells) and blood TFR cells are less suppressive (compared toLN TFR cells). Additionally, in memory experiments, the TFH to TFR ratiois skewed towards the TFH cell. The combination of these cell intrinsicand extrinsic differences allow circulating memory TFH cells to quicklyrespond to antigen and activate B cells in lymph nodes after a secondaryexposure to antigen. We show in parabiosis experiments that the CXCR5+population of the non-immunized parabiotic mouse is dominated by bloodderived CXCR5+ cells after immunization, demonstrating that theblood-derived CXCR5+ population controls the GC response more potentlythan newly generated LN CXCR5 cells. We hypothesize this is a keymechanism by which quick and efficient secondary responses are generatedupon re-exposure to antigen in distant tissues. Understanding themechanisms of TFH and TFR cell memory should provide insights intostrategies for developing long-lasting B cell memory to vaccines and forcontrolling pathogenic B cell responses.

Experimental Procedures

Mice. 6-8 week old mice were used for all experiments. WT C57BL/6,CIITA^(−/−) /tax-DTR (CD11c-DTR), Ighm^(−/−) (mMT) and Actin^(CFP) micewere purchased from The Jackson Laboratory (Bar Harbor, Me.). CD28^(−/−)mice were generated as described (Shahinian et al., 1993). Actin^(CFP)Fox^(GFP) mice were generated by crossing Actin^(CFP) mice withFox-IRES-GFP mice (Bettelli et al., 2006). For CD11c-DTR experiments,bone marrow chimeras were made by irradiating WT recipient mice with 600rads twice, separated by 3 hours, and adoptively transferring 7×10⁶ bonemarrow cells. Mice were used after 8 weeks of reconstitution, andreconstitution was confirmed by flow cytometry. Mice were injected with1 mg diphtheria toxin (Sigma) intraperitoneally every 2 days starting atday 0 after immunization and harvested on day 7. In some experiments WTmice were injected intraperitoneally with 25 mg/kg FTY720 (CaymanChemical) every 2 days starting at day 2 after immunization.Alternatively, mice were injected with the FTY720 3 or 6 hours beforeharvesting organs. Thoracic duct cannulation was performed as describedpreviously (Massberg et al., 2007). All mice were used according to theHarvard Medical School Standing Committee on Animals and NationalInstitutes of Animal Healthcare Guidelines. Animal protocols wereapproved by the Harvard Medical School Standing Committee on Animals.

Immunizations

For NP-OVA immunizations, 100 μg NP₁₈-OVA (Biosearch Technologies) in a1:1 H37RA CFA (DIFCO) emulsion was injected subcutaneously on the mouseflanks. Seven days later mice were euthanized and inguinal lymph nodes(dLN) were harvested, blood was collected via cardiac puncture with a 1cc syringe and immune cells were isolated by sucrose densitycentrifugation using Lymphocyte Separation Media (LSM).

Bone Marrow Derived Dendritic Cells

Bone marrow derived dendritic cells (BMDCs) were made by culturing totalbone marrow cells in the presence of 30 ng/ml GM-CSF (Peprotech) for 7days. During the last 24 hours, 20 ng/ml of LPS (sigma) and 20 μg/ml ofNP-OVA were added to the cultures. Cells were harvested, washedextensively and counted on an Accuri cytometer. 1×10⁶ cells weresubcutaneously injected in 100 ul PBS on the flank of WT mice. 5 dayslater the inguinal lymph node and blood were harvested and analyzed forTFH and TFR cells.

Flow Cytometry

Cells from lymphoid organs were isolated and resuspended in stainingbuffer (PBS containing 1% fetal calf serum and 2 mM EDTA) and stainedwith directly labeled antibodies from Biolegend against CD4 (RM4-5),ICOS (15F9), CD19 (6D5), CD69 (H1.2F3) CD11c (N418), MHC II(M5/114.15.2), IL17A (TC11-18H10.1), IL2 (JES6-5H4), IFNg (XMG1.2), IL4(11B11), TNFα (MP6-XT22), CD138 (281-2), from eBioscience against FoxP3(FJK-165), Bcl6 (mGI191E), IL21 (FFA21), from BD bioscience against GL7,Ki67 (B56) and IgG1 (A85-1), and from Abcam, goat anti-GFP. For CXCR5staining, biotinylated anti-CXCR5 (2G8, BD Biosciences) was usedfollowed by streptavidin-brilliant violet 421 (Biolegend). Forintracellular staining of transcription factors and Ki67, the FoxP3fix/perm kit was used (eBioscience) after surface staining wasaccomplished. For intracellular cytokine staining, cells were incubatedwith 1 μg/ml ionomycin (Sigma) and 500 ng/ml PMA (Sigma) in the presenceof Golgistop (BD biosciences) for 4 hours prior to staining. All flowcytometry was analyzed with an LSR II (BD biosciences) using standardfilter sets.

Adoptive Transfers

For CXCR5⁺ T cell transfers, 20 Actin^(CFP) Fox^(GFP) mice wereimmunized with NP-OVA subcutaneously as described above, and 7 dayslater dLN or blood was collected. 2×10⁵ CFP⁺CD4⁺ICOS⁺CXCR5⁺ cells wereadoptively transferred to CD28^(−/−) or WT mice that were then immunizedsubcutaneously with NP-OVA. For memory experiments, recipients wereimmunized after 30 days. 7 days later organs were harvested. Skin fromthe immunization site was digested with 500 U/ml collagenase(Worthington Biochemical). Transferred cells were identified by eitherCFP expression or intracellularly with an anti-GFP polyclonal antibody(Abcam).

Parabiosis

For parabiosis, age- and sex-matched Actin-^(CFP) Fox-^(GFP) mice andC57Bl/6 mice were anesthetized by ketamine/xylazine (i.p.) andsurgically joined at the olecranons and knees as described previously(Massberg et al., 2007). Briefly, the lateral sides of each mouse wereshaved and wiped with depilatory cream, and incisions were made alongthe lateral aspect of each mouse from the olecranon to the knee. Thesubcutaneous fascia was bluntly dissected to expose loose ventral anddorsal skin flaps for connection. Mice were first joined together at theolecranons and knees by a double ligature using 4-0 braided silk suture.Next, the ventral and dorsal skin flaps overlying the olecranon and kneeregion were joined by continuous 6-0 braided silk suture. Finally, theremaining dorsal and ventral skin flaps between the olecranons and kneeswere connected by staples. Parabiotic mice were separated after 19 daysby reversal of the above procedure. Mice were immunized with NP-OVAsubcutaneously on the non-joined side 6 days later. Chimerism amongdifferent leukocyte populations was calculated as the average of thepercent of CFP+ cells in the WT mouse in the non-draining lymph nodes.

In Vitro Suppression Assay

All populations were sorted to 98% purity on a FACS Aria using standarddetectors. Cells were counted on an Accuri cytometer (BD biosciences) bygating live cells only. For TFH stimulation assays, 2×10⁴ dLN or bloodCD4⁺ICOS⁺CXCR5⁺CD19⁻GITR⁻ TFH cells were plated with 5×10⁴ CD19⁺ B cellsor 2×10⁴ CD3⁻CD19⁻CD11c⁺ DCs, all from dLNs of WT mice immunized withNP-OVA 7 days previously. For DCs, dLNs were digested with 500 U/mlCollagenase I (Worthington Biochemical) before isolation. TFH plus B orDCs were cultured in the presence of 2 μg/ml soluble anti-CD3 (2C11,BioXcell) and 5 μg/ml anti-IgM (Jackson Immunoresearch) for 5-6 days.Cells were then harvested and stained for flow cytometry. Intracellularexpression of Bcl6 and Ki67 were used as readouts for TFH cellactivation and functional potential. Intracellular expression of IgG1was used as a readout for B cell class switch recombination. For TFRsuppression assays, 1×10⁴ CD4⁺ICOS⁺CXCR5⁺CD19⁻GITR⁺ TFR cells fromeither the dLN or blood of WT mice immunized with NP-OVA 7 dayspreviously were added to the wells of TFH stimulation assays. Cells wereharvested 5-6 days later.

Confocal Microscopy

Confocal microscopy was performed as described previously (Sage et al.,2013). Briefly, organs were harvested and embedded and frozen in OCTmedium (Tissue-Tek). 10 um sections were cut, fixed and stained usingthe FoxP3 kit (ebioscience). Samples were imaged using an Olympusconfocal microscope using standard configurations.

Statistical Analysis

Unpaired Student's t test was used for all comparisons, data representedas mean+/−SD or SE are shown. P values <0.05 were consideredstatistically significant. * P<0.05, ** P<0.005, *** P<0.0005.

(G) Quantification of lymph node and blood TFR cells from plots in (F).Total CD4⁺FoxP3⁺CD19⁻ cells “Total FoxP3+” are included as controls.(H-J) CXCR5 (H), ICOS (I), Ki67 (J) expression on lymph node and bloodTFR cells gated as in (F). Data are means+/−standard error with 5 miceper group. Data are representative of at least 3 independentexperiments.

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Example 4. Comparison of TFR and Treg Gene Expression Signatures

TFR (CD4+ICOS+CXCR5+GITR+CD19−) cells were sorted from the lymph nodesof 20 pooled, WT or PD-1 deficient mice 7 days after subcutaneousimmunization with NP-OVA in CFA. Similarly, CD4+CXCR5−FoxP3+ Tregs weresorted from the lymph nodes of unimmunized FoxP3 reporter mice. Eachsort was performed in triplicate. RNA was isolated using standardprocedures, was amplified and reverse transcription was performed.Samples were run on mouse affymetrix gene expression arrays. Data wasthen normalized. Top 100 hits were determined by comparing WT TFR toTregs and ranked by signal-to-noise. PD-1 deficient TFR cells, whichhave enhanced suppressive capacity, are added as an additional signatureof TFR cells. Results are shown in FIG. 23.

Example 5. Surface Receptors Differentially Expressed by Human Blood TFRCells

Fresh human peripheral blood mononuclear cells were isolated by sucrosedensity centrifugation from normal human blood. Cells were surfacestained with anti-CD4, anti-CXCR5, and anti-CD19. Cells were then platedon 96-well plates that contained antibodies to surface receptors in thescreen. Cells were then fixed, permeabilized and intracellularly stainedwith anti-FoxP3. Populations were analyzed by flow cytometry as CD4(CD4+CXCR5−FoxP3−CD19−), Treg (CD4+CXCR5−FoxP3+CD19−), TFH(CD4+CXCR5+FoxP3−CD19−) and TFR (CD4+CXCR5+FoxP3+CD19−). Top 56 hits (ofTFR versus TFH cell with a fold increase of at least 1.2) are shown inFIG. 24, sorted on mean fluorescence intensity on TFR cells (value).

Example 6. Surface Receptors Differentially Expressed by Human Blood TFHCells

Fresh human peripheral blood mononuclear cells were isolated by sucrosedensity centrifugation from normal human blood. Cells were surfacestained with anti-CD4, anti-CXCR5, and anti-CD19. Cells were then platedon 96-well plates that contained antibodies to surface receptors in thescreen. Cells were then fixed, permeabilized and intracellularly stainedwith anti-FoxP3. Populations were analyzed by flow cytometry as CD4(CD4+CXCR5−FoxP3−CD19−), Treg (CD4+CXCR5− FoxP3+CD19−), TFH(CD4+CXCR5+FoxP3−CD19−) and TFR (CD4+CXCR5+FoxP3+CD19−). Top 19 hits(TFH versus TFR) are shown in FIG. 25, sorted by fold increase ofexpression on TFH versus TFR cell. Value indicates expression (in meanfluorescence intensity) on TFR cells.

Example 7. Blockade of the PD-1 Pathway can Heighten AntibodyStimulating Capacity of TFH Cells

Murine CD4+ICOS+CXCR5+GITR−CD19− TFH cells were sorted from the lymphnodes of WT mice (that were immunized subcutaneously with NP-OVA in CFA7 days previously) on an Aria cell sorter. TFH cells were plated withCD19+ B cells (isolated from lymph nodes of mice immunized with NP-OVA 7days previously) in the presence of 5 ug/ml anti-IgM and 2 ug/mlanti-CD3 for 6 days. Anti-PDL1 was also added to some wells. After 6days, cells from cultures were harvested, surface stained with anti-CD4,-CD19, -GL7, and -I-A followed by fixation/permeabilization, andintracellularly stained with anti-IgG1. Cells were analyzed by flowcytometric analysis. B cells were identified by surface expression ofCD19 and IA. Ig class switched B cells were identified by coexpressionof GL7 and IgG1. Results are shown in FIG. 26.

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. All other published references, documents,manuscripts and scientific literature cited herein are herebyincorporated by reference.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims. It should also be understood thatthe embodiments described herein are not mutually exclusive and thatfeatures from the various embodiments may be combined in whole or inpart in accordance with the invention.

1-27. (canceled)
 28. A composition comprising a population ofT-follicular regulatory (TFR) cells isolated from the peripheral bloodof a subject wherein the composition is enriched for TFR cells. 29-100.(canceled)
 101. The composition of claim 28, comprising about 50% TFRcells or more of the total T-cells present in the composition.
 102. Thecomposition of claim 28, comprising about 90% TFR cells or more of thetotal T-cells present in the composition.
 103. The composition of claim28, wherein the TFR cells have been expanded.
 104. The composition ofclaim 28, wherein the TFR cells have been expanded in the presence of aPD-1 receptor antagonist or PD-1 ligand inhibitor.
 105. A method ofmodulating an immune response in a subject comprising administering tothe subject, an effective amount of a composition of claim
 28. 106. Themethod of claim 105, wherein after administration of the composition,the ratio of TFR cells to TFH cells found in the peripheral blood of thesubject is higher as compared to the ratio of TFR cells to TFH cells inthe patient prior to the administration of the composition.
 107. Themethod of claim 105, wherein an immune response in the subject issuppressed.
 108. The method of claim 105, wherein the subject has adisease or disorder selected from graft versus host disease (GVHD),organ rejection, autoimmune disease, and side effects of gene therapy.109. A method of preparing a composition enriched for TFR cellscomprising the steps of: a) obtaining an initial population of cellscomprising T cells; b) enriching for TFR cells from the populationwherein the TFR cells are sorted based on surface markers ofCD4⁺CXCR5⁺ICOS⁺ and at least one surface marker selected from: GITR⁺,CD25^(hi), CD162, CD27, CD95, CD9, CD43, CD50, CD45RB, CD102, CD61,CD58, CD196, CD38, CD31, CD15, CD25, CD13, CD66a/c/e, CD11b CD63, CD32,CD97, HLA-HQ, CD150, Siglec-9, Integrinβ7, CD71, CD180, CD218a, CD193,CD235ab, CD35, CD140a, CD158b, CD33, CD210, HLA-G, CD167a, CD119,CX3CR1, CD146, HLA-DR, CD85, CD172b, SSEA-1, CD49c, CD170, CD66b, andCD86.
 110. The method of claim 109, wherein the initial population ofcells is isolated from the peripheral blood, tissues or organs of one ormore subjects.
 111. The method of claim 109, wherein the TFR cellscomprise about 50% or more of the total T cells present in thecomposition.
 112. The method of claim 109, wherein the TFR cellscomprise about 90% or more of the total T cells present in thecomposition.
 113. The method of claim 109, wherein the TFR cells furthercomprising the step of expanding the TFR cells of step (b).
 114. Themethod of claim 113, wherein the TFR cells have been expanded in thepresence of a PD-1 receptor antagonist or PD-1 ligand inhibitor.